49 results on '"Agalakova NI"'
Search Results
2. Monoclonal antibody to an endogenous bufadienolide, marinobufagenin, reverses preeclampsia-induced Na/K-ATPase inhibition and lowers blood pressure in NaCl-sensitive hypertension.
- Author
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Fedorova OV, Simbirtsev AS, Kolodkin NI, Kotov AY, Agalakova NI, Kashkin VA, Tapilskaya NI, Bzhelyansky A, Reznik VA, Frolova EV, Nikitina ER, Budny GV, Longo DL, Lakatta EG, Bagrov AY, Fedorova, Olga V, Simbirtsev, Andrey S, Kolodkin, Nikolai I, Kotov, Alexander Y, and Agalakova, Natalia I
- Published
- 2008
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3. Brain ouabain stimulates peripheral marinobufagenin via angiotensin II signalling in NaCl-loaded Dahl-S rats.
- Author
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Fedorova OV, Agalakova NI, Talan MI, Lakatta EG, Bagrov AY, Fedorova, Olga V, Agalakova, Natalia I, Talan, Mark I, Lakatta, Edward G, and Bagrov, Alexei Y
- Published
- 2005
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4. Antibody to marinobufagenin lowers blood pressure in pregnant rats on a high NaCl intake.
- Author
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Fedorova OV, Kolodkin NI, Agalakova NI, Namikas AR, Bzhelyansky A, St-Louis J, Lakatta EG, Bagrov AY, Fedorova, Olga V, Kolodkin, Nikolai I, Agalakova, Natalia I, Namikas, Alexandra R, Bzhelyansky, Anton, St-Louis, Jean, Lakatta, Edward G, and Bagrov, Alexei Y
- Published
- 2005
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5. Antibody to Endogenous Cardiotonic Steroid Reverses Vascular Fibrosis and Restores Vasorelaxation in Chronic Kidney Disease.
- Author
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Agalakova NI, Mikhailova EV, Ershov IA, Nadei OV, Pyankov AA, Galagoudza MM, Adair CD, Romanova IV, and Bagrov AY
- Subjects
- Animals, Male, Rats, Aorta drug effects, Aorta metabolism, Antibodies pharmacology, Nephrectomy, Nitroprusside pharmacology, Proto-Oncogene Protein c-fli-1 metabolism, Collagen Type I metabolism, Endothelin-1 metabolism, Renal Insufficiency, Chronic drug therapy, Renal Insufficiency, Chronic metabolism, Fibrosis, Vasodilation drug effects, Rats, Sprague-Dawley, Bufanolides pharmacology
- Abstract
Marinobufagenin (MBG) is implicated in chronic kidney disease, where it removes Fli1-induced inhibition of the collagen-1. We hypothesized that (i) in nephrectomized rats, aortic fibrosis develops due to elevated plasma MBG and inhibited Fli1, and (ii) that the antibody to MBG reduces collagen-1 and improves vasodilatation. A partial nephrectomy was performed in male Sprague-Dawley rats. Sham-operated animals comprised the control group. At 5 weeks following nephrectomy, rats were administered the vehicle ( n = 8), or the anti-MBG antibody ( n = 8). Isolated aortic rings were tested for their responsiveness to sodium nitroprusside following endothelin-1-induced constriction. In nephrectomized rats, there was an increase in the intensity of collagen staining in the aortic wall vs. the controls. In antibody-treated rats, the structure of bundles of collagen fibers had ordered organization. Western blots of the aorta had lower levels of Fli1 (arbitrary units, 1 ± 0.05 vs. 0.2 ± 0.01; p < 0.001) and greater collagen-1 (arbitrary units, 1 ± 0.01 vs. 9 ± 0.4; p < 0.001) vs. the control group. Administration of the MBG antibody to rats reversed the effect of the nephrectomy on Fli1 and collagen-1 proteins. Aortic rings pretreated with endothelin-1 exhibited 50% relaxation following the addition of sodium nitroprusside (EC
50 = 0.28 μmol/L). The responsiveness of the aortic rings obtained from nephrectomized rats was markedly reduced (EC50 = 3.5 mol/L) compared to the control rings. Treatment of rats with the antibody restored vasorelaxation. Thus, the anti-MBG antibody counteracts the Fli1-collagen-1 system and reduces aortic fibrosis.- Published
- 2024
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6. Chloroquine and Chemotherapeutic Compounds in Experimental Cancer Treatment.
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Agalakova NI
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- Animals, Humans, Phosphatidylinositol 3-Kinases, Therapies, Investigational, Hydroxychloroquine pharmacology, Hydroxychloroquine therapeutic use, Antineoplastic Agents, Alkylating, Cytotoxins, Chloroquine pharmacology, Chloroquine therapeutic use, Neoplasms drug therapy
- Abstract
Chloroquine (CQ) and its derivate hydroxychloroquine (HCQ), the compounds with recognized ability to suppress autophagy, have been tested in experimental works and in clinical trials as adjuvant therapy for the treatment of tumors of different origin to increase the efficacy of cytotoxic agents. Such a strategy can be effective in overcoming the resistance of cancer cells to standard chemotherapy or anti-angiogenic therapy. This review presents the results of the combined application of CQ/HCQ with conventional chemotherapy drugs (doxorubicin, paclitaxel, platinum-based compounds, gemcitabine, tyrosine kinases and PI3K/Akt/mTOR inhibitors, and other agents) for the treatment of different malignancies obtained in experiments on cultured cancer cells, animal xenografts models, and in a few clinical trials. The effects of such an approach on the viability of cancer cells or tumor growth, as well as autophagy-dependent and -independent molecular mechanisms underlying cellular responses of cancer cells to CQ/HCQ, are summarized. Although the majority of experimental in vitro and in vivo studies have shown that CQ/HCQ can effectively sensitize cancer cells to cytotoxic agents and increase the potential of chemotherapy, the results of clinical trials are often inconsistent. Nevertheless, the pharmacological suppression of autophagy remains a promising tool for increasing the efficacy of standard chemotherapy, and the development of more specific inhibitors is required.
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- 2024
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7. Optimal Reference Genes for RT-qPCR Experiments in Hippocampus and Cortex of Rats Chronically Exposed to Excessive Fluoride.
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Nadei OV and Agalakova NI
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- Rats, Animals, Male, Real-Time Polymerase Chain Reaction methods, Rats, Wistar, Gene Expression genetics, Hippocampus, Reference Standards, Fluorides, Gene Expression Profiling methods
- Abstract
Normalization of the quantitative real-time PCR (RT-qPCR) data to the stably expressed reference genes is critically important for obtaining reliable results. However, all previous studies focused on F
- toxicity for brain tissues used a single, non-validated reference gene, what might be a cause of contradictory or false results. The present study was designed to analyze the expression of a series of reference genes to select optimal ones for RT-qPCR analysis in cortex and hippocampus of rats chronically exposed to excessive fluoride (F- ) amounts. Six-week-old male Wistar rats randomly assigned to four groups consumed regular tap water with 0.4 (control), 5, 20, and 50 ppm F- (NaF) for 12 months. The expression of six genes (Gapdh, Pgk1, Eef1a1, Ppia, Tbp, Helz) was compared by RT-qPCR in brain tissues from control and F- -exposed animals. The stability of candidate reference genes was evaluated by coefficient of variation (CV) analysis and RefFinder online program summarizing the results of four well-acknowledged statistical methods (Delta-Ct, BestKeeper, NormFinder, and GeNorm). In spite of some discrepancies in gene ranking between these algorisms, Pgk1, Eef1a1, and Ppia were found to be most valid in cortex, while Ppia, Eef1a1, and Helz showed the greatest expression stability in hippocampus. Tbp and Helz were identified as the least stable genes in cortex, whereas Gapdh and Tbp are unsuitable for hippocampus. These data indicate that reliable mRNA quantification in the cortex and hippocampus of F- -poisoned rats is possible using normalization to geometric mean of Pgk1+Eef1a1 or Ppia+Eef1a1 expression, respectively., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)- Published
- 2024
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8. Fli1 and Tissue Fibrosis in Various Diseases.
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Mikhailova EV, Romanova IV, Bagrov AY, and Agalakova NI
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- Humans, Fibrosis, Oncogene Proteins, Fusion genetics, Proto-Oncogene Protein c-fli-1 genetics, Proto-Oncogene Protein c-fli-1 metabolism, RNA-Binding Protein EWS genetics, Sarcoma, Ewing genetics, Scleroderma, Systemic genetics, Scleroderma, Systemic pathology
- Abstract
Being initially described as a factor of virally-induced leukemias, Fli1 (Friend leukemia integration 1) has attracted considerable interest lately due to its role in both healthy physiology and a variety of pathological conditions. Over the past few years, Fli1 has been found to be one of the crucial regulators of normal hematopoiesis, vasculogenesis, and immune response. However, abnormal expression of Fli1 due to genetic predisposition, epigenetic reprogramming (modifications), or environmental factors is associated with a few diseases of different etiology. Fli1 hyperexpression leads to malignant transformation of cells and progression of cancers such as Ewing's sarcoma. Deficiency in Fli1 is implicated in the development of systemic sclerosis and hypertensive disorders, which are often accompanied by pronounced fibrosis in different organs. This review summarizes the initial findings and the most recent advances in defining the role of Fli1 in diseases of different origin with emphasis on its pro-fibrotic potential.
- Published
- 2023
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9. Silencing of Fli1 Gene Mimics Effects of Preeclampsia and Induces Collagen Synthesis in Human Umbilical Arteries.
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Agalakova NI, Reznik VA, Ershov IA, Lupanova EA, Nadei OV, Ivanov DO, David Adair C, and Bagrov AY
- Subjects
- Animals, Collagen Type I metabolism, Female, Humans, Pregnancy, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Rats, Sodium-Potassium-Exchanging ATPase metabolism, Umbilical Arteries, Bufanolides metabolism, Pre-Eclampsia genetics, Pre-Eclampsia metabolism, Proto-Oncogene Protein c-fli-1 genetics
- Abstract
Background: Previously we demonstrated that in patients with preeclampsia elevated levels of endogenous Na/K-ATPase inhibitor, marinobufagenin, cause inhibition of Friend leukemia virus integration 1 (Fli1), a negative regulator of collagen-1 synthesis. We hypothesized that in vitro silencing of Fli1 in healthy human umbilical arteries would be associated with an increase in collagen-1 output, similar to the effect of preeclampsia in rat and human tissues., Methods: The isolated segments of healthy human umbilical arteries were tested for sensitivity to MBG and Fli1 silencing with Fli1 siRNA or control siRNA., Results: Following 24-hour incubation of arteries with nanomolar concentrations of marinobufagenin, Fli1 expression was inhibited 5-fold (P < 0.001), and synthesis of collagen-1 increased 3 times (P < 0.01). Twenty-four-hour incubation of umbilical artery fragments with Fli1 siRNA caused a dramatic decrease of Fli1 (7-fold; P < 0.001) and cytoplasmic PKC δ (4-fold; P < 0.001) expression in comparison to control siRNA or untreated control, followed by elevation in procollagen (3-fold; P < 0.001) and collagen-1 (3-fold; P < 0.001) levels in vascular tissue., Conclusions: Our results show that after silencing the Fli1 gene in healthy human umbilical arteries a new phenotype emerges which is typical for preeclampsia and is associated with vascular fibrosis., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Journal of Hypertension, Ltd. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2022
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10. Canrenone Restores Vasorelaxation Impaired by Marinobufagenin in Human Preeclampsia.
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Agalakova NI, Grigorova YN, Ershov IA, Reznik VA, Mikhailova EV, Nadei OV, Samuilovskaya L, Romanova LA, Adair CD, Romanova IV, and Bagrov AY
- Subjects
- Animals, Canrenone, Collagen Type I metabolism, Female, Fibrosis, Humans, Mineralocorticoid Receptor Antagonists pharmacology, Pregnancy, Rats, Sodium-Potassium-Exchanging ATPase metabolism, Vasodilation, Bufanolides pharmacology, Pre-Eclampsia drug therapy, Pre-Eclampsia pathology
- Abstract
Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Recently, we demonstrated that (i) MBG induces fibrosis in rat tissues via a mechanism involving Fli1, a negative regulator of collagen-1 synthesis, and (ii) MBG sensitive Na/K-ATPase inhibition is reversed by mineralocorticoid antagonists. We hypothesized that in human PE elevated MBG level is associated with the development of fibrosis of the umbilical arteries and that this fibrosis can be attenuated by canrenone. Fifteen patients with PE (mean BP = 118 ± 4 mmHg; 34 ± 2 years; 38 ± 0.3 weeks gest. age) and twelve gestational age-matched normal pregnant subjects (mean BP = 92 ± 2 mmHg; 34 ± 1 years; 39 ± 0.2 weeks gest. age) were enrolled in the study. PE was associated with a higher plasma MBG level, with a four-fold decrease in Fli1 level and a three-fold increase in collagen-1 level in the PE umbilical arteries vs. those from the normal subjects (p < 0.01). Isolated rings of umbilical arteries from the subjects with PE exhibited impaired responses to the relaxant effect of sodium nitroprusside vs. control vessels (EC50 = 141 nmol/L vs. EC50 = 0.9 nmol/L; p < 0.001). The effects of PE on Fli1 and collagen-1 were blocked by the in vitro treatment of umbilical arteries by 10 μmol/L canrenone. Similar results were obtained for umbilical arteries pretreated with MBG. These data demonstrate that elevated MBG level is implicated in the development of the fibrosis of umbilical arteries in PE, and that this could be blocked by mineralocorticoid antagonists.
- Published
- 2022
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11. Differential protein expression of mitogen-activated protein kinases in erythrocytes and liver of lamprey Lampetra fluviatilis on the course of prespawning starvation.
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Khvorova IA, Nadei OV, and Agalakova NI
- Subjects
- Animals, Erythrocytes enzymology, Female, Fish Proteins blood, Lampreys blood, Male, Mitogen-Activated Protein Kinases blood, Reproduction, Seasons, Starvation blood, Starvation enzymology, Subcellular Fractions enzymology, Fish Proteins metabolism, Lampreys metabolism, Liver enzymology, Mitogen-Activated Protein Kinases metabolism
- Abstract
The study was designed to identify the types of mitogen-activated protein kinases (MAPKs) in erythrocytes and liver tissues of river lamprey Lampetra fluviatilis and monitor the changes in protein expression levels of found enzymes on the course of prespawning starvation (from November to the end of May). Immunoreactivity of the native and phosphorylated forms of ERK1/2, JNK and p38 was examined in the cytosolic and membrane cell fractions. Both lamprey erythrocytes and liver were found to highly express ERK1/2 and JNK, whereas only trace amounts of p38 were revealed in hepatic tissues. ERK1/2 was identified in cytosolic and membrane fractions, whereas JNK and p38 were predominantly cytosolic enzymes. Total cellular amounts of ERK1/2 and phospho-ERK1/2 in both erythrocytes and liver tissues appeared to be relatively stable on the course of prespawning starvation. However, before spawning ERK1/2 translocated from cytosol to membranes, with partial decline of its cytoplasmic expression being compensated by increases in membrane-bound pool. Immunoreactivity of cytoplasmic JNK, phospho-JNK and p38 were stable from November to March, but sharply decreased before spawning exhibiting almost negligible levels in May, which suggests the depletion of their cellular fractions. Most probably, ERK1/2 plays more important role in mediating adaptive responses of erythrocytes and liver tissues to conditions of natural starvation and maintenance of cell viability before spawning and death of animals in May., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2022
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12. Preeclampsia: Cardiotonic Steroids, Fibrosis, Fli1 and Hint to Carcinogenesis.
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Agalakova NI, Kolodkin NI, Adair CD, Trashkov AP, and Bagrov AY
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- Animals, Antibodies, Monoclonal therapeutic use, Bufanolides immunology, Bufanolides metabolism, Female, Fibrosis, Humans, Immunotherapy methods, Pregnancy, Proto-Oncogene Mas, Proto-Oncogene Protein c-fli-1 antagonists & inhibitors, Proto-Oncogene Protein c-fli-1 metabolism, Signal Transduction drug effects, Signal Transduction immunology, Sodium-Potassium-Exchanging ATPase metabolism, Arteries pathology, Carcinogenesis drug effects, Cardiac Glycosides pharmacology, Cardiac Glycosides therapeutic use, Pre-Eclampsia drug therapy, Pre-Eclampsia metabolism
- Abstract
Despite prophylaxis and attempts to select a therapy, the frequency of preeclampsia does not decrease and it still takes the leading position in the structure of maternal mortality and morbidity worldwide. In this review, we present a new theory of the etiology and pathogenesis of preeclampsia that is based on the interaction of Na/K-ATPase and its endogenous ligands including marinobufagenin. The signaling pathway of marinobufagenin involves an inhibition of transcriptional factor Fli1, a negative regulator of collagen synthesis, followed by the deposition of collagen in the vascular tissues and altered vascular functions. Moreover, in vitro and in vivo neutralization of marinobufagenin is associated with the restoration of Fli1. The inverse relationship between marinobufagenin and Fli1 opens new possibilities in the treatment of cancer; as Fli1 is a proto-oncogene, a hypothesis on the suppression of Fli1 by cardiotonic steroids as a potential anti-tumor therapeutic strategy is discussed as well. We propose a novel therapy of preeclampsia that is based on immunoneutralization of the marinobufagenin by monoclonal antibodies, which is capable of impairing marinobufagenin-Na/K-ATPase interactions.
- Published
- 2021
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13. Cognitive Decline of Rats with Chronic Fluorosis Is Associated with Alterations in Hippocampal Calpain Signaling.
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Nadei OV, Khvorova IA, and Agalakova NI
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- Animals, Brain-Derived Neurotrophic Factor metabolism, Hippocampus metabolism, Maze Learning, Nuclear Proteins, Rats, Rats, Sprague-Dawley, Calpain metabolism, Cognitive Dysfunction chemically induced
- Abstract
The study was designed to evaluate an influence of excessive fluoride (F
- ) intake on cognitive capacities of adult rats and on proteins of memory-related calpain signaling in hippocampus. Control animals were given water with natural F- content of 0.4 ppm; rats from other groups consumed the same water supplemented with 5, 20, and 50 ppm F- (as NaF) for 12 months. The efficiency of learning and memory formation was evaluated by novel object recognition (NOR) and Morris water maze tests. The expression of enzymes of calpain-1 and calpain-2 signaling in hippocampus was detected by Western blotting. Excessive F- consumption had moderate impact on short-term memory, but impaired spatial learning and long-term memory of animals. Intoxication of rats with 5-50 ppm F- led to stimulation of calpain-1 in hippocampal cells and its translocation from cytosol to membranes, accompanied by activation of GTPase RhoA. Exposure to 20-50 ppm F- resulted in proteolytic cleavage of phosphatase PHLPP1 and increased expression of phospho-ERK1/2 kinase with insignificant decline of total ERK1/2 activity. In contrast, F- did not change the expression of calpain-2 and its substrates-phosphatase PTEN and kinase mTOR. However, F- intake led to downregulation of cAMP-response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF). Thus, altered expression of calpain-1 and its downstream effectors at a background of stable activity of calpain-2 indicates overstimulation of signaling pathways of early LTP phase and disrupted link between early and late LTP phases, most probably due to altered activity of transcriptional and neurotrophic factors.- Published
- 2020
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14. Antibody against Na/K-ATPase Inhibitor Lowers Blood Pressure and Increases Vascular Fli1 in Experimental Preeclampsia.
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Agalakova NI, Reznik VA, Nadei OV, Ershov IA, Rassokha OS, Vasyutina ML, Ivanov DO, Adair CD, Galagudza MM, and Bagrov AY
- Subjects
- Animals, Bufanolides metabolism, Disease Models, Animal, Female, Fibrosis, Pre-Eclampsia enzymology, Pre-Eclampsia pathology, Pre-Eclampsia physiopathology, Pregnancy, Rats, Sprague-Dawley, Sodium Chloride, Dietary, Umbilical Arteries enzymology, Umbilical Arteries pathology, Umbilical Arteries physiopathology, Up-Regulation, Antibodies pharmacology, Antihypertensive Agents pharmacology, Blood Pressure drug effects, Bufanolides antagonists & inhibitors, Pre-Eclampsia prevention & control, Proto-Oncogene Protein c-fli-1 metabolism, Sodium-Potassium-Exchanging ATPase metabolism, Umbilical Arteries drug effects
- Abstract
Background: Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). We demonstrated that MBG induces fibrosis via mechanism involving inhibition of Fli1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis. We hypothesized that PE blockade of increased MBG with antibody would lessen the fibrosis of umbilical arteries and lower the blood pressure in rats with PE., Methods: We tested 36 pregnant Sprague-Dawley rats in which 12 were made hypertensive by 1.8% Na supplementation (days 6-19 of gestation), 12 pregnant rats served controls. At day 19, PE rats received one intraperitoneal injection of polyclonal anti-MBG-4 antibody (0.5 ug/ml) for 4 hours., Results: PE was associated with higher blood pressure (117 ± 2 vs. 107 ± 2 mm Hg; P < 0.01), plasma MBG levels (1.54 ± 0.34 vs. 0.49 ± 0.11 nmol/L; P < 0.01), protein excretion (26 vs. 12 mg/24 hours), sFlt-1 (3-fold), decrease in Fli1 (7-fold) and increase in collagen-1 in aorta (4-fold) vs. control rats (all P < 0.01). In 12 rats treated with polyclonal anti-MBG-4 antibody blood pressure dropped (93 ± 3 mm Hg) and Fli1 was decreased much less (2-fold; P < 0.01 vs. nontreated rats)., Conclusions: These results demonstrate that in experimental PE elevated MBG level is implicated in umbilical fibrosis via suppression of Fli1., (© American Journal of Hypertension, Ltd 2019. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
- Published
- 2020
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15. Inorganic fluoride and functions of brain.
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Agalakova NI and Nadei OV
- Subjects
- Central Nervous System drug effects, Humans, Brain drug effects, Fluorides toxicity, Hazardous Substances toxicity
- Abstract
Although actively disputed and questioned, it has been proposed that chronic exposure to inorganic fluoride (F
- ) is toxic for brain. The major question for this review was whether an excessive F- intake is causally related to adverse neurological and cognitive health conditions in human beings and animals. The paper systematically and critically summarizes the findings of the studies showing positive associations between F- intoxication and various intellectual defects, as well as of those which attempted to clarify the nature of F- neurotoxicity. Many works provide support for a link between pre- and postnatal F- exposure and structural and functional changes in the central nervous system responsible for neurological and cognitive disorders. The mechanisms suggested to underlie F- neurotoxicity include the disturbances in synaptic transmission and synaptic plasticity, premature death of neurons, altered activities of components of intracellular signaling cascades, impaired protein synthesis, deficit of neurotrophic and transcriptional factors, oxidative stress, metabolic changes, inflammatory processes. However, the majority of works have been performed on laboratory rodents using such F- doses which are never exist in the nature even in the regions of endemic fluorosis. Thus, this kind of treatment is hardly comparable with human exposure even taking into account the higher rate of F- clearance in animals. Of special importance are the data collected on humans chronically consuming excessive F- doses in the regions of endemic fluorosis or contacting with toxic F- compounds at industrial sites, but those works are scarce and often criticized due to low quality. New, expertly performed studies with repeated exposure assessment in independent populations are needed to prove an ability of F- to impair neurological and intellectual development of human beings and to understand the molecular mechanisms implicated in F- -induced neurotoxicity.- Published
- 2020
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16. Endogenous Bufadienolides, Fibrosis and Preeclampsia.
- Author
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Reznik VA, Kashkin VA, Agalakova NI, Adair CD, and Bagrov AY
- Abstract
Frequency of preeclampsia has no tendency to decrease, and it still takes the leading position in the structure of maternal mortality and morbidity worldwide. In this review, we present the "fibrotic concept" of the etiology and pathogenesis of preeclampsia which involves system consisting of Na/K-ATPase and its endogenous ligands including marinobufagenin. New therapy of preeclampsia includes modulation of the Na/K-ATPase system by immunoneutralization of the marinobufagenin and use of mineralocorticoid antagonists which are capable to impair marinobufagenin-Na/K-ATPase interactions., Competing Interests: All authors declare no conflicts of interest., (Copyright © 2019 Vitaliy A. Reznik et al.)
- Published
- 2019
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17. Cardiotonic Steroids Induce Vascular Fibrosis Via Pressure-Independent Mechanism in NaCl-Loaded Diabetic Rats.
- Author
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Fedorova OV, Fadeev AV, Grigorova YN, Marshall CA, Zernetkina V, Kolodkin NI, Agalakova NI, Konradi AO, Lakatta EG, and Bagrov AY
- Subjects
- Animals, Aorta metabolism, Aorta pathology, Aorta physiopathology, Aortic Diseases metabolism, Aortic Diseases pathology, Aortic Diseases physiopathology, Blood Pressure, Collagen metabolism, Diabetes Mellitus, Experimental metabolism, Diabetes Mellitus, Experimental physiopathology, Diabetes Mellitus, Type 2 metabolism, Diabetes Mellitus, Type 2 physiopathology, Erythrocytes drug effects, Erythrocytes enzymology, Fibrosis, Hypertension metabolism, Hypertension physiopathology, Male, Proto-Oncogene Protein c-fli-1 metabolism, Rats, Wistar, Signal Transduction, Sodium-Potassium-Exchanging ATPase blood, Transforming Growth Factor beta1 metabolism, Aorta drug effects, Aortic Diseases chemically induced, Bufanolides pharmacology, Diabetes Mellitus, Experimental complications, Diabetes Mellitus, Type 2 complications, Hypertension complications, Sodium Chloride, Vascular Remodeling drug effects, Vascular Stiffness drug effects
- Abstract
Endogenous cardiotonic steroid, marinobufagenin (MBG), induces Fli1-dependent tissue fibrosis. We hypothesized that an increase in MBG initiates the development of aortic fibrosis in salt-loaded rats with type 2 diabetes mellitus (DM2) via pressure-independent mechanism. DM2 was induced by a single intraperitoneal administration of 65 mg/kg streptozotocin to neonatal (4-5 days) male Wistar rats. Eight-week-old DM2 rats received water or 1.8% NaCl (DM-NaCl) solution for 4 weeks (n = 16); half of DM-NaCl rats were treated with anti-MBG monoclonal antibody (mAb) (DM-NaCl-AB) during week 4 of salt loading; control intact rats received water (n = 8/group). Blood pressure, MBG, erythrocyte Na/K-ATPase activity, aortic weights, levels of fibrosis markers (Fli1, protein kinase Cδ, transforming growth factor-β1, receptors of the transforming growth factor beta5, fibronectin, collagen-1), and sensitivity of the aortic explants to the vasorelaxant effect of sodium nitroprusside were assessed. No changes in systolic blood pressure were observed while erythrocyte Na/K-ATPase was inhibited by 30%, plasma MBG was doubled, and aortic markers of fibrosis became elevated in DM-NaCl rats versus control. Treatment of DM-NaCl rats with anti-MBG mAb activated Na/K-ATPase, prevented increases in aortic weights, and the levels of fibrosis markers returned to the control levels. The responsiveness of the aortic rings from DM-NaCl rats to the relaxant effect of sodium nitroprusside was reduced (half maximal effective concentration (EC50) = 29 nmol/L) versus control rings (EC50 = 7 nmol/L) and was restored by anti-MBG mAb (EC50 = 9 nmol/L). Our results suggest that in salt-loaded diabetic rats, MBG stimulates aortic collagen synthesis in a pressure-independent fashion and that 2 profibrotic mechanisms, Fli1 dependent and transforming growth factor-β dependent, underlie its effects.
- Published
- 2019
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18. Antibody to Marinobufagenin Reverses Placenta-Induced Fibrosis of Umbilical Arteries in Preeclampsia.
- Author
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Fedorova OV, Ishkaraeva VV, Grigorova YN, Reznik VA, Kolodkin NI, Zazerskaya IE, Zernetkina V, Agalakova NI, Tapilskaya NI, Adair CD, Lakatta EG, and Bagrov AY
- Subjects
- Adult, Animals, Antibodies immunology, Blood Pressure, Bufanolides blood, Case-Control Studies, Collagen Type I metabolism, Erythrocytes enzymology, Female, Fibrosis, Gestational Age, Humans, Immunotherapy, Microfilament Proteins antagonists & inhibitors, Microfilament Proteins metabolism, Pre-Eclampsia immunology, Pre-Eclampsia pathology, Pregnancy, Rats, Receptors, Cytoplasmic and Nuclear antagonists & inhibitors, Receptors, Cytoplasmic and Nuclear metabolism, Sodium-Potassium-Exchanging ATPase metabolism, Trans-Activators, Umbilical Arteries pathology, Antibodies therapeutic use, Bufanolides immunology, Placenta metabolism, Pre-Eclampsia drug therapy, Umbilical Arteries metabolism
- Abstract
Background: Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Immunoneutralization of heightened MBG by Digibind, a digoxin antibody, reduces blood pressure (BP) in patients with PE, and anti-MBG monoclonal antibody lessens BP in a rat model of PE. Recently, we demonstrated that MBG induces fibrosis in cardiovascular tissues via a mechanism involving inhibition of Fli-1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis., Objectives and Methods: We hypothesized that in PE, elevated placental MBG levels are associated with development of fibrosis in umbilical arteries. Eleven patients with PE (mean BP 124 ± 4 mmHg; age 29 ± 2 years; 39 weeks gest. age) and 10 gestational age-matched normal pregnant subjects (mean BP 92 ± 2 mmHg; controls) were enrolled in the clinical study., Results: PE was associated with a higher placental (0.04 ± 0.01 vs. 0.49 ± 0.11 pmol/g; p < 0.01) and plasma MBG (0.5 ± 0.1 vs. 1.6 ± 0.5 nmol/L; p < 0.01), lower Na/K-ATPase activity in erythrocytes (2.7 ± 0.2 vs. 1.5 ± 0.2 µmol Pi/mL/hr; p < 0.01), 9-fold decrease of Fli-1 level and 2.5-fold increase of collagen-1 in placentae ( p < 0.01) vs. control. Incubation of umbilical arteries from control patients with 1 nmol/L MBG was associated with four-fold decrease in Fli-1 level and two-fold increase in collagen-1 level vs. those incubated with placebo ( p < 0.01), i.e., physiological concentration of MBG mimicked effect of PE in vitro. Collagen-1 abundance in umbilical arteries from PE patients was 4-fold higher than in control arteries, and this PE-associated fibrosis was reversed by monoclonal anti-MBG antibody ex vivo., Conclusion: These results demonstrate that elevated placental MBG level is implicated in the development of fibrosis of the placenta and umbilical arteries in PE.
- Published
- 2018
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19. Understanding quasi-apoptosis of the most numerous enucleated components of blood needs detailed molecular autopsy.
- Author
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Gusev GP, Govekar R, Gadewal N, and Agalakova NI
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- Cell Survival, Humans, Aging blood, Apoptosis physiology, Cell Death, Erythrocyte Aging physiology, Erythrocytes physiology
- Abstract
Erythrocytes are the most numerous cells in human body and their function of oxygen transport is pivotal to human physiology. However, being enucleated, they are often referred to as a sac of molecules and their cellularity is challenged. Interestingly, their programmed death stands a testimony to their cell-hood. They are capable of self-execution after a defined life span by both cell-specific mechanism and that resembling the cytoplasmic events in apoptosis of nucleated cells. Since the execution process lacks the nuclear and mitochondrial events in apoptosis, it has been referred to as quasi-apoptosis or eryptosis. Several studies on molecular mechanisms underlying death of erythrocytes have been reported. The data has generated a non-cohesive sketch of the process. The lacunae in the present knowledge need to be filled to gain deeper insight into the mechanism of physiological ageing and death of erythrocytes, as well as the effect of age of organism on RBCs survival. This would entail how the most numerous cells in the human body die and enable a better understanding of signaling mechanisms of their senescence and premature eryptosis observed in individuals of advanced age., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
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20. Apoptotic death in erythrocytes of lamprey Lampetra fluviatilis induced by ionomycin and tert-butyl hydroperoxide.
- Author
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Agalakova NI, Ivanova TI, Gusev GP, Nazarenkova AV, and Sufiyeva DA
- Subjects
- Animals, Biomarkers blood, Biomarkers metabolism, Cell Size, Cell Survival drug effects, Erythrocytes cytology, Erythrocytes metabolism, Female, Kinetics, Lampreys, Male, Membrane Potential, Mitochondrial drug effects, Oxidative Stress drug effects, Rivers, Russia, Apoptosis drug effects, Calcium Ionophores toxicity, Erythrocytes drug effects, Ionomycin toxicity, Oxidants toxicity, Water Pollutants, Chemical toxicity, tert-Butylhydroperoxide toxicity
- Abstract
The work examined the effects of Ca
2+ overload and oxidative damage on erythrocytes of river lamprey Lampetra fluvialtilis. The cells were incubated for 3h with 0.1-5μM Ca2+ ionophore ionomycin in combination with 2.5mM Ca2+ and 10-100μM pro-oxidant agent tert-butyl hydroperoxide (tBHP). The sensitivity of lamprey RBCs to studied compounds was evaluated by the kinetics of their death. Both toxicants induced dose- and time dependent phosphatidylserine (PS) externalization (annexin V-FITC labeling) and loss of membrane integrity (propidium iodide uptake). Highest doses of ionomycin (1-2μM) increased the number of PS-exposed erythrocytes to 7-9% within 3h, while 100μM tBHP produced up to 50% of annexin V-FITC-positive cells. Caspase inhibitor Boc-D-FMK (50μM), calpain inhibitor PD150606 (10μM) and broad protease inhibitor leupeptin (200μM) did not prevent ionomycin-induced PS externalization, whereas tBHP-triggered apoptosis was blunted by Boc-D-FMK. tBHP-dependent death of lamprey erythrocytes was accompanied by the decrease in relative cell size, loss of cell viability, activation of caspases 9 and 3/7, and loss of mitochondrial membrane potential, but all these processes were partially attenuated by Boc-D-FMK. None of examined death-associated events were observed in ionomycin-treated erythrocytes except activation of caspase-9. Incubation with ionomycin did not alter intracellular K+ and Na+ content, while exposure to tBHP resulted in 80% loss of K+ and 2.8-fold accumulation of Na+ . Thus, lamprey erythrocytes appear to be more susceptible to oxidative damage. Ca2+ overload does not activate the cytosolic death pathways in these cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)- Published
- 2017
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21. ATP-consuming processes in hepatocytes of river lamprey Lampetra fluviatilis on the course of prespawning starvation.
- Author
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Agalakova NI, Brailovskaya IV, Konovalova SA, Korotkov SM, Lavrova EA, and Nikiforov AA
- Subjects
- Adenosine Triphosphate metabolism, Alanine Transaminase blood, Animals, Aspartate Aminotransferases blood, Coumaric Acids pharmacology, Cycloheximide pharmacology, Female, Gluconeogenesis drug effects, Hepatocytes drug effects, Hepatocytes metabolism, Lampreys physiology, Membrane Potential, Mitochondrial drug effects, Mitochondria, Liver metabolism, Oviposition physiology, Phenylpyruvic Acids pharmacology, Protein Synthesis Inhibitors pharmacology, Rivers, Seasons, Sodium-Potassium-Exchanging ATPase metabolism, Starvation metabolism, Lampreys metabolism
- Abstract
The work was performed to establish which of the major ATP-consuming processes is the most important for surviving of hepatocytes of female lampreys on the course of prespawning starvation. The requirements of protein synthesis and Na(+)-K(+)-ATPase for ATP in the cells were monitored by the changes in mitochondrial membrane potential (MMP) in the presence of corresponding inhibitors from the peak of metabolic depression (January-February) to the time of recovery from it (March-April) and spawning (May). Integrity of lamprey liver cells was estimated by catalytic activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in blood plasma. In January-February, the share of ATP necessary for protein synthesis was 20-22%, whereas before spawning it decreased to 8-11%. Functioning of Na(+)-K(+)-pump required 22% of cellular ATP at the peak of metabolic depression, but 38% and 62% of ATP in March-April and May, respectively. Progression of prespawning period was accompanied by 3.75- and 1.6-fold rise of ALT and AST activities in blood plasma, respectively, whereas de Ritis coefficient decreased from 2.51±0.34 to 0.81±0.08, what indicates severe damage of hepatocyte membranes. Thus, the adaptive strategy of lamprey hepatocytes to develop metabolic depression under conditions of energy limitation is the selective production of proteins necessary for spawning, most probably vitellogenins. As spawning approaches, the maintenance of transmembrane ion gradients, membrane potential and cell volume to prevent premature cell death becomes the priority cell function., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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22. Transient activation of protein kinase C contributes to fluoride-induced apoptosis of rat erythrocytes.
- Author
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Agalakova NI and Gusev GP
- Subjects
- Animals, Apoptosis drug effects, Calcium metabolism, Cells, Cultured, Erythrocytes metabolism, Isoenzymes metabolism, Male, Rats, Rats, Wistar, Erythrocytes drug effects, Protein Kinase C metabolism, Sodium Fluoride toxicity
- Abstract
Role of PKC in fluoride-induced apoptosis of rat erythrocytes was studied in vitro and in vivo. Treatment of erythrocytes with 5 mM NaF for 1–24 h caused progressive accumulation of cytosolic Ca2+ and PS exposure at outer membrane surface. After 1 h, these processes were suppressed by PKC inhibitors staurosporine, GF 109203X and chelerythrine, but increased by PKC activator PMA. Following 24 h, NaF-induced Ca2+ uptake and PS externalization were partly prevented by PMA or staurosporine, but not by GF 109293X and chelerythrine. Application of PP inhibitor OA augmented NaF-induced cell responses within 1 h, but not after 24 h. Incubation of erythrocytes with 0.1–10 mM NaF for 1 h produced a dose-dependent PKCa translocation from cytosol to membranes with appearance of active PKM fragment. 24 h NaF exposure led to complete loss of cytosolic PKCa and proteolysis of membrane PKCa. Besides, NaF weakly stimulated membrane PKCf, although its subcellular distribution was not altered. Thus, transient PKCa activation/translocation positively contributes to NaF-induced apoptosis in vitro. Consumption of 2–20 ppm fluoride by the rats for 12 months also induced dose-dependent PKCa translocation to membranes and activation of membrane PKCf, what indicates that PKC stimulation is an important physiological mechanism of fluoride toxicity.
- Published
- 2013
23. Excessive fluoride consumption leads to accelerated death of erythrocytes and anemia in rats.
- Author
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Agalakova NI and Gusev GP
- Subjects
- Adenosine Triphosphate biosynthesis, Animals, Calcium metabolism, Cytosol metabolism, Dose-Response Relationship, Drug, Erythrocytes metabolism, Fluorides administration & dosage, Male, Rats, Rats, Wistar, Reactive Oxygen Species metabolism, Anemia chemically induced, Cell Death drug effects, Erythrocytes drug effects, Fluorides toxicity
- Abstract
The present study was performed to evaluate an overall effect of long-term consumption of excessive fluoride (F) amounts by rats on their erythrocytes. The animals were administered regular drinking water (0.4 ppm F) or the same water supplemented with 2, 10, and 20 ppm F (as NaF) for 12 months. Chronic exposure of the rats to increasing F doses induced a progressive rise of the plasma F concentration accompanied by a dose-dependent fall of hematocrit and decrease in the mean erythrocyte volume. Consumption of 10 and 20 ppm F resulted in appearance of morphologically abnormal cells (stomatocytes and echinocytes) in the peripheral blood. Rise of the water F concentration to 20 ppm F led to significant increase in the number of phosphatidylserine-exposing erythrocytes, although suppression of cell viability was revealed in all three groups of F-poisoned rats. A compensatory enhanced release of reticulocytes was not sufficient to compensate for erythrocyte loss. Dose-dependent accumulation of free cytosolic Ca(2+) appears to be a major pathophysiological process underlying the development of F-induced death processes in rat erythrocytes. In addition, 10 and 20 ppm F induced ATP depletion and generation of peroxides in erythrocytes, whereas superoxide and glutathione levels were not altered. Thus, long-term intoxication of the rats with F triggers premature death of their erythrocytes due to intrinsic death-associated biochemical defects and development of anemia.
- Published
- 2013
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24. Fluoride induces oxidative stress and ATP depletion in the rat erythrocytes in vitro.
- Author
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Agalakova NI and Gusev GP
- Subjects
- Animals, Cells, Cultured, Erythrocytes metabolism, Glutathione metabolism, Male, Peroxides metabolism, Rats, Rats, Wistar, Superoxides metabolism, Adenosine Triphosphate metabolism, Cariostatic Agents toxicity, Erythrocytes drug effects, Oxidative Stress, Sodium Fluoride toxicity
- Abstract
The present study was designed to examine an ability of inorganic fluoride (F) to induce oxidative stress and energy depletion in the rat erythrocytes in vitro. Accumulation of ROS and alterations in glutathione (GSH) and ATP contents were estimated in the cells incubated with 0.1-10mM NaF for 1, 5 and 24h. Exposure of the rat erythrocytes to NaF was accompanied by progressive accumulation of peroxides, while superoxide (O(2)(-)) production was insignificant. Intracellular GSH content was reduced following 5-h incubation, but considerably elevated after 24h, although GSH/GSSG ratio decreased in both cases. ATP concentration in the NaF-treated cell exhibited a dose- and time-dependent decline, diminishing to extremely low levels within 24h. Thus, exposure of the rat erythrocytes to NaF leads to impairment of the cellular antioxidant system and severe energy depletion, the latter probably being the primary toxic effect., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2012
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25. Fluoride-induced death of rat erythrocytes in vitro.
- Author
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Agalakova NI and Gusev GP
- Subjects
- Animals, Calcium metabolism, Cell Membrane drug effects, Cell Membrane metabolism, Cell Size drug effects, Cell Survival drug effects, Cells, Cultured, Ceramides metabolism, Erythrocytes metabolism, Erythrocytes pathology, Hemoglobins metabolism, Hemolysis, Male, Rats, Rats, Wistar, Cariostatic Agents toxicity, Cytotoxins toxicity, Erythrocytes drug effects, Sodium Fluoride toxicity
- Abstract
Although fluoride (F) in low concentrations is essential for teeth and bone development, its excessive consumption causes numerous deleterious abnormalities in cellular metabolism and physiology often leading to cell death. The present study was performed to establish the toxic F effects inducing the death of rat erythrocytes in vitro. The cells were cultured in the presence of 0.5-16 mM NaF for 1, 5 and 24 h. The progression of erythrocyte death was monitored by cell viability (calcein assay), membrane integrity (hemolysis assay), alterations in the cell morphology (light microscopy) and size (flow cytometry forward scatter), plasma membrane scrambling (annexin V binding). To elucidate the molecular mechanisms underlying F-induced cell death, the cytosolic Ca2+ activity (Fluo-3 fluorescence) and ceramide formation (binding of FITC-labeled antibodies) were determined. Exposure of the rat erythrocytes to NaF considerably suppressed their viability and caused partial cell hemolysis within 24 h. The cells underwent dramatic morphological alterations resulted in appearance of shrunken echinocytes after 1h and swollen spherocytes within 24 h. The development of NaF-induced erythrocyte death was accompanied by progressive PS externalization at the outer cell membrane, ∼45% of the cells were annexin V-positive in response to 16 mM NaF within 24 h with a small cell population exhibiting necrotic features. The cell death was preceded by considerable accumulation of the free cytosolic Ca2+, with statistically significant increase in the number of Fluo-3-positive erythrocytes observed as early as during 1-h incubation with 0.5 mM NaF. NaF also induced moderate ceramide formation. Overall, exposure of the rat erythrocytes to NaF triggers rapid progression of their death in a dose- and time-dependent manner, with appearance of apoptotic cells after 1 and 5 h and transition to necrosis within 24 h. An increase in intracellular [Ca2+] appears to be crucial mechanism implicated in development of NaF-induced apoptosis in rat erythrocytes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
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26. [Effect of inorganic fluorine on living organisms of different phylogenetic level].
- Author
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Agalakova NI and Gusev GP
- Subjects
- Animals, Fluoridation, Fluorine deficiency, Fluorosis, Dental etiology, Fluorosis, Dental metabolism, Humans, Phylogeny, Tissue Distribution, Fluorine metabolism, Fluorine toxicity
- Abstract
The presented review summarizes literature data on pathways of the inorganic fluoride intake into the plant, animal, and human organisms, on its metabolism, distribution, and accumulation in the organism, forms of fluoride in biological tissues, toxic effects of fluoride on physiological and reproductive functions of living organisms of various phylogenetic groups, as well as clinical symptoms of deficient and excessive fluoride intake into the human organism.
- Published
- 2011
27. Regulation of K-Cl cotransport in erythrocytes of frog Rana temporaria by commonly used protein kinase and protein phosphatase inhibitors.
- Author
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Gusev GP and Agalakova NI
- Subjects
- Animals, Benzophenanthridines pharmacology, Biological Transport drug effects, Biological Transport physiology, Erythrocytes drug effects, Fluorides pharmacology, Genistein pharmacology, Okadaic Acid pharmacology, Ouabain pharmacology, Phosphoprotein Phosphatases antagonists & inhibitors, Potassium metabolism, Protein Kinase C antagonists & inhibitors, Protein-Tyrosine Kinases antagonists & inhibitors, Quercetin pharmacology, Rana temporaria, Staurosporine pharmacology, Symporters drug effects, Tyrphostins pharmacology, K Cl- Cotransporters, Enzyme Inhibitors pharmacology, Erythrocytes metabolism, Phosphoprotein Phosphatases physiology, Protein Kinase C physiology, Protein Kinase Inhibitors pharmacology, Protein-Tyrosine Kinases physiology, Symporters physiology
- Abstract
Recently (Agalakova and Gusev in J Comp Physiol 179:443-450, 2009), we demonstrated that the activity of K-Cl cotransport (KCC) in frog red blood cells is inhibited under stimulation of protein kinase C (PKC) with phorbol ester PMA (12-myristate-13-acetate). Present work was performed to uncover possible implication of protein kinases and protein phosphatases (PPs) in the regulation of baseline and volume-dependent KCC activity in these cells. K+ influx was estimated as 86Rb uptake by the cells in isotonic or hypotonic media in the presence of ouabain, K+ efflux was determined as the difference between K+ loss by the cells incubated in parallel in isotonic or hypotonic K(+)-free Cl(-)- and NO(3)(-)-media. Swelling of the cells in hypotonic medium was accompanied by approximately 50% activation of Cl-dependent K+ influx and efflux. Protein tyrosine kinase (PTK) inhibitor genistein (0.1 mM) stably and considerably (up to 89%) suppressed both baseline and volume-dependent KCC activity in each direction. Other PTK blockers (tyrphostin 23 and quercetin) had no influence on KCC activity in frog erythrocytes. PKC inhibitor chelerythrine (20 microM) and both PP inhibitors, fluoride (5 mM) and okadaic acid (1 microM), reduced KCC activity by 25-70%. Neither basal nor swelling-activated KCC in frog erythrocytes was affected by PKC inhibitor staurosporine (1 microM). Based on the previous and present results, we can suggest that the main role in the maintenance of basal and volume-dependent KCC activity in frog erythrocytes belongs to PTKs and PPs, whereas PKC is a negative regulator of this ion system.
- Published
- 2010
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28. Endogenous cardiotonic steroids and differential patterns of sodium pump inhibition in NaCl-loaded salt-sensitive and normotensive rats.
- Author
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Bagrov AY, Agalakova NI, Kashkin VA, and Fedorova OV
- Subjects
- Animals, Bufanolides pharmacology, Cardiac Glycosides pharmacology, Cyclic GMP urine, Kidney drug effects, Male, Muscle, Smooth, Vascular drug effects, Ouabain pharmacology, Rats, Rats, Inbred Dahl, Rats, Sprague-Dawley, Sodium metabolism, Bufanolides metabolism, Cardiac Glycosides metabolism, Sodium Chloride, Dietary administration & dosage, Sodium-Potassium-Exchanging ATPase antagonists & inhibitors
- Abstract
Background: Endogenous sodium pump inhibitors promote sodium excretion in normotensives and contribute to vasoconstriction in NaCl-sensitive hypertension. Marinobufagenin (MBG), an endogenous bufadienolide inhibitor of alpha-1 sodium pump, contributes to hypertension in Dahl salt-sensitive rats (DS). We hypothesized that in NaCl-loaded DS and normotensive Sprague-Dawley rats (S-D), MBG would elicit different patterns of sodium pump inhibition., Methods: We compared systolic blood pressure (SBP), renal sodium excretion, activity of the sodium pump in aorta and renal medulla, and levels of MBG, atrial natriuretic peptide (ANP), and cyclic guanosine monophosphate (cGMP) in salt-loaded DS and S-D (20% NaCl, 2.5 ml/kg, intraperitoneally)., Results: NaCl loading produced sustained elevations in renal MBG excretion in both DS (2.41 +/- 0.24 vs. 0.79 +/- 0.08 pmol/h/kg, P < 0.01) and S-D (1.97 +/- 0.37 vs. 0.60 +/- 0.07 pmol/h/kg, P < 0.01) vs. that at baseline (n = 10 for each group). In NaCl-loaded DS, SBP rose by 18 mm Hg (P < 0.01) and aortic sodium pump was inhibited by 22% (P < 0.05 vs. control), while in S-D, SBP and activity of aortic sodium pump did not change. NaCl-loaded S-D excreted twice as much sodium as DS; in S-D, renal sodium pump was inhibited by 24% vs. 14% inhibition in DS (P < 0.05). NaCl loading elicited increases in plasma ANP and in renal cGMP excretion in S-D but not in DS., Conclusions: Our present observations demonstrate that in NaCl-loaded S-D and DS, a comparable MBG response is associated with preferential inhibition of the sodium pump in the kidney and in vascular smooth muscle, respectively, resulting in an adaptive natriuresis in S-D but sodium retention and pressor response in DS.
- Published
- 2009
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29. Effects of phorbol 12-myristate 13-acetate on potassium transport in the red blood cells of frog Rana temporaria.
- Author
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Agalakova NI and Gusev GP
- Subjects
- Animals, Ion Transport drug effects, Protein Kinase C metabolism, Rubidium Radioisotopes, Scintillation Counting, Sodium-Potassium-Exchanging ATPase metabolism, Symporters metabolism, Tetradecanoylphorbol Acetate pharmacology, Erythrocytes metabolism, Potassium metabolism, Rana temporaria metabolism, Tetradecanoylphorbol Acetate analogs & derivatives
- Abstract
Phorbol 12-myristate 13-acetate (PMA), a stimulator of PKC, was examined for its influence on K(+) ((86)Rb) influx in the frog erythrocyte. PMA, 0.1 microM, was found to accelerate ouabain-sensitive K(+) influx, which was suppressed by 73% with 1 mM amiloride, indicating secondary activation of the Na(+)-K(+)-pump due to stimulation of Na(+ )/H(+) exchange. PMA-induced stimulation of the sodium pump was completely inhibited with 1 microM staurosporine and by approximately 50% with 20 microM chelerythrine. In contrast to Na(+)-K(+)-pump, an activity of Cl(-)-dependent K(+) transport (K-Cl cotransport, KCC), calculated as the difference between K(+) influxes in Cl(-) and NO(3) (-)-media, was substantially decreased under the influence of PMA. Staurosporine fully restored the PMA-induced inhibition of KCC, whereas chelerythrine did not exert any influence. Osmotic swelling of the frog erythrocytes was accompanied by approximately twofold stimulation of KCC. Swelling-activated KCC was inhibited by approximately 50 and approximately 83% in the presence of PMA and genistein, respectively, but not chelerythrine. Exposure of the frog erythrocytes to 5 mM fluoride (F(-)) also reduced the KCC activity in isotonic and hypotonic media, with maximal suppression of K(+) influx in both media being observed upon simultaneous addition of PMA and F(-). Furosemide and [(dihydronindenyl)oxy] alkanoic acid inhibited the K(+) influx in both the media by approximately 50-60%. The results obtained show both the direct and indirect effects of PMA on the K(+) transport in frog erythrocytes and a complicated picture of KCC regulation in frog erythrocytes with involvement of PKC, tyrosine kinase and protein phosphatase.
- Published
- 2009
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30. Transport of lithium across the lamprey (Lampetra fluviatilis) erythrocyte membrane.
- Author
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Gusev GP, Agalakova NI, and Ivanova TI
- Subjects
- Amiloride pharmacology, Animals, Antiporters physiology, Biological Transport, Active, Cantharidin pharmacology, Cations, Monovalent, Erythrocyte Membrane drug effects, Hydrogen-Ion Concentration, Erythrocyte Membrane physiology, Lampreys physiology, Lithium metabolism, Sodium metabolism
- Abstract
Lithium, capable of replacing Na+ in various membrane transport processes, was used to investigate Na+ transport pathways across the lamprey erythrocytes membrane. The values of Li+ influxes have ranged from 8 to 24 mmol/l cells/h. Intracellular accumulation of Li+ was associated with loss of cellular Na+, the value of which was less than the value of Li+ influx. Both Li+ influx and Na+ efflux were partially inhibited by amiloride. The amiloride-sensitive Li+ influx was considerably stimulated by hyperosmotic cell shrinkage. The treatment of lamprey erythrocytes with blockers of protein phosphatases (fluoride and cantharidin) also resulted in a considerable increase in Li+ accumulation within the cells. No significant difference was observed between the values of Li+ and Na+ (22Na) influxes measured in red cells incubated simultaneously in isotonic LiCl and NaCl media (9.2 +/- 2.1 and 7.8 +/- 1.3 mmol/l cells/h, respectively). In hypo- and hypertonic media, however, the rate of Na+ influx in lamprey erythrocytes was approximately twice higher as compared to the rate of Li+ influx, what was determined by the difference in the amiloride-sensitive components. In acidified lamprey erythrocytes (intracellular pH 6.0) Li+ and Na+ influxes were considerably increased due to activation of amiloride-sensitive Na+/H+ (Li+/H+) exchange mechanism, although the activity of Na+/H+ exchange was much greater than that of Li+/H+ exchange. The data obtained confirm the hypothesis on the presence of two amiloride-sensitive systems of Na+ transport in the lamprey red blood cells.
- Published
- 2008
31. Intrahippocampal microinjection of an exquisitely low dose of ouabain mimics NaCl loading and stimulates a bufadienolide Na/K-ATPase inhibitor.
- Author
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Fedorova OV, Zhuravin IA, Agalakova NI, Yamova LA, Talan MI, Lakatta EG, and Bagrov AY
- Subjects
- Animals, Dose-Response Relationship, Drug, Male, Microinjections, Ouabain administration & dosage, Rats, Bufanolides pharmacology, Enzyme Inhibitors pharmacology, Hippocampus, Ouabain pharmacology, Sodium Chloride administration & dosage, Sodium-Potassium-Exchanging ATPase antagonists & inhibitors
- Abstract
Background: Brain endogenous ouabain (EOU) raises blood pressure (BP) via an angiotensin II (ATII)-sensitive pathway in NaCl-loaded Dahl salt-sensitive rats (DSS). Brain EOU activates central and adrenocortical renin-angiotensin systems, and stimulates marinobufagenin, a vasoconstrictor and natriuretic inhibitor of sodium pump., Methods: We studied effects of acute NaCl loading (17 mmol/kg NaCl, intraperitoneally) on levels of EOU and marinobufagenin in several brain areas in DSS. We then studied effects of intrahippocampal administration of very-low-dose ouabain (60 pg) on EOU, marinobufagenin, BP, sodium excretion, and sodium-pump activity in the aorta and renal medulla in the absence and presence of anti-marinobufagenin and anti-ouabain antibodies, and losartan., Results: NaCl loading of DSS induced transient increases of EOU in the hippocampus and amygdala (15 min; 300%), supraoptical nucleus of hypothalamus (SON) (30 min, 230%) and pituitary (30 min; 85%), and ATII elevation in the SON (30 min). Intrahippocampal administration of ouabain (60 pg) stimulated ATII in the SON, produced natriuresis, 40 mmHg rise in BP, inhibition of sodium-pump in the renal medulla (19.6%) and aorta (25%), and a two-fold increase in renal marinobufagenin excretion. Pretreatment of rats with anti-marinobufagenin antibody prevented ouabain-induced pressor and natriuretic responses and sodium-pump inhibition. Pressor responses to ouabain were also prevented by losartan (intravenously) and by administration of anti-ouabain antibody into the SON., Conclusions: NaCl loading of DSS induces a cascade of events, triggered by brain EOU and ATII. Intrahippocampal administration of a low-dose ouabain mimics effects of NaCl loading and stimulates marinobufagenin, which produces natriuresis, and inhibits the vascular sodium-pump, inducing an increase in BP.
- Published
- 2007
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32. ANP differentially modulates marinobufagenin-induced sodium pump inhibition in kidney and aorta.
- Author
-
Fedorova OV, Agalakova NI, Morrell CH, Lakatta EG, and Bagrov AY
- Subjects
- Animals, Aorta metabolism, Enzyme Inhibitors pharmacology, In Vitro Techniques, Kidney Medulla metabolism, Male, Phosphorylation drug effects, Rats, Rats, Sprague-Dawley, Sodium-Potassium-Exchanging ATPase antagonists & inhibitors, Sodium-Potassium-Exchanging ATPase metabolism, Vasodilator Agents pharmacology, Aorta drug effects, Atrial Natriuretic Factor pharmacology, Bufanolides pharmacology, Kidney Medulla drug effects, Sodium-Potassium-Exchanging ATPase drug effects
- Abstract
NaCl loading and plasma volume expansion stimulate 2 natriuretic systems, vasoconstrictor, digitalis-like Na/K-ATPase inhibitors and vasorelaxant ANP peptides. Several hormones, including ANP, regulate activity of the Na/K-ATPase by modulation of its phosphorylation state. We studied effects of ANP on Na/K-ATPase phosphorylation and inhibition by an endogenous sodium pump ligand, marinobufagenin, in the aorta and renal medulla from male Sprague-Dawley rats. Marinobufagenin dose-dependently inhibited the Na/K-ATPase in renal and vascular membranes at the level of higher (nanomolar) and lower affinity (micromolar) binding sites. Marinobufagenin (1 nmol/L) inhibited Na/K-ATPase in aortic sarcolemma (18%) and in renal medulla (19%). prepro-ANP 104 to 123 (ppANP) and alpha-human ANP ([alpha-hANP] both 1 nmol/L) potentiated marinobufagenin-induced Na/K-ATPase inhibition in the kidney, but reversed the effect of marinobufagenin in the aorta. Similarly, ppANP and alpha-hANP modulated the sodium pump (ouabain-sensitive (86)Rb uptake) inhibitory effects of marinobufagenin in the aorta and renal medulla. In renal medulla, ppANP and alpha-hANP induced alpha-1 Na/K-ATPase phosphorylation, whereas in aorta, both peptides dephosphorylated Na/K-ATPase. The effect of ppANP on Na/K-ATPase phosphorylation and inhibition was mimicked by a protein kinase G activator, 8-Br-PET-cGMP (10 micromol/L), and prevented by a protein kinase G inhibitor, KT5823 (60 nmol/L). Our results suggest that alpha-1 Na/K-ATPase inhibition by marinobufagenin in the kidney is enhanced via Na/K-ATPase phosphorylation by ANP, whereas in the aorta, ANP exerts an opposite effect. The concurrent production of a vasorelaxant, ANP, and a vasoconstrictor, marinobufagenin, potentiate each other's natriuretic effects, but ANP peptides may offset the deleterious vasoconstrictor effect of marinobufagenin.
- Published
- 2006
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33. Activation of sodium transport in rat erythrocytes by inhibition of protein phosphatases 1 and 2A.
- Author
-
Ivanova TI, Agalakova NI, and Gusev GP
- Subjects
- Amiloride pharmacology, Animals, Cantharidin pharmacology, Erythrocytes enzymology, Ion Transport drug effects, Male, Marine Toxins, Okadaic Acid pharmacology, Oxazoles pharmacology, Phosphoprotein Phosphatases metabolism, Rats, Rats, Wistar, Sodium Fluoride pharmacology, Sodium-Potassium-Chloride Symporters metabolism, Solute Carrier Family 12, Member 1, Enzyme Inhibitors pharmacology, Erythrocytes metabolism, Phosphoprotein Phosphatases antagonists & inhibitors, Sodium-Hydrogen Exchangers metabolism
- Abstract
Four structurally different protein phosphatases (PPs) inhibitors - fluoride, calyculin A, okadaic acid and cantharidin--were tested for their ability to modulate unidirectional Na(+) influx in rat red blood cells. Erythrocytes were incubated at 37 degrees C in isotonic and hypertonic media containing 1 mM ouabain and (22)Na in the absence or presence of PP inhibitors. Exposure of the cells to 20 mM fluoride or 50 nM calyculin A for 1 h under isosmotic conditions caused a significant stimulation of Na(+) influx, whereas addition of 200 microM cantharidin or 100 nM okadaic acid had no effect. After 2 h of treatment, however, all these PPs blockers significantly enhanced Na(+) transport in rat erythrocytes. Selective inhibitors of PP-1 and PP-2A types, calyculin A, cantharidin and okadaic acid, produced similar ( approximately 1.2-1.4-fold) stimulatory effects on Na(+) influx in the cells. Activation of Na(+) influx was unchanged with increasing calyculin A concentration from 50 to 200 nM. No additive stimulation of Na(+) influx was observed when the cells were treated with combination of 20 mM fluoride and 50 nM calyculin A. Na(+) influx induced by PPs blockers was inhibited by 1 mM amiloride and 200 muM bumetanide approximately in the equal extent, indicating the involvement of Na(+)/H(+) exchange and Na-K-2Cl cotransport in sodium transport through rat erythrocytes membrane. Activation of Na(+) transport in the cells induced by calyculin A and fluoride was associated with increase of intracellular Na(+) content. Shrinkage of the rat erythrocytes resulted in 2-fold activation of Na(+) influx. All tested PPs inhibitors additionally activated the Na(+) influx by 70-100% above basal shrinkage-induced level. Amiloride and bumetanide have diminished both the shrinkage-induced and PPs-inhibitors-induced Na(+) influxes. Thus, our observations clearly indicate that activities of Na(+)/H(+) exchanger and Na-K-2Cl cotransporter in rat erythrocytes are regulated by protein phosphatases and stimulated when protein dephosphorylation is inhibited.
- Published
- 2006
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34. Coordinated shifts in Na/K-ATPase isoforms and their endogenous ligands during cardiac hypertrophy and failure in NaCl-sensitive hypertension.
- Author
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Fedorova OV, Talan MI, Agalakova NI, Lakatta EG, and Bagrov AY
- Subjects
- Animals, Bufanolides blood, Cardiac Output, Low etiology, Cardiac Output, Low metabolism, Cardiomegaly etiology, Cardiomegaly metabolism, Enzyme Inhibitors blood, Hypertension complications, Isoenzymes metabolism, Ligands, Male, Ouabain blood, Rats, Rats, Inbred Dahl, Cardiac Output, Low enzymology, Cardiomegaly enzymology, Hypertension physiopathology, Sodium Chloride pharmacology, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
Objectives: NaCl loading of Dahl salt-sensitive rats (DS) stimulates marinobufagenin (MBG), an alpha1 Na/K-ATPase (NKA) isoform ligand. Cardiac function depends on NKA, which is regulated in part by endogenous digitalis-like ligands. Our goal was to study whether changes occur in MBG and endogenous ouabain (EO) production during cardiac remodelling in hypertensive DS, and whether these are associated with changes in myocardial NKA isoforms and sensitivity to MBG and ouabain., Methods: Changes in MBG and EO levels, changes in myocardial NKA isoform composition, and sensitivity to endogenous ligands during development of cardiac hypertrophy and the transition to heart failure were studied in DS rats with an 8% NaCl intake., Results: The animals developed compensated left ventricular hypertrophy after 4 weeks, which progressed to heart failure at 9-12 weeks. The hypertrophic stage was associated with increased plasma MBG levels (mean +/- SEM of 1.22 +/- 0.22 versus 0.31 +/- 0.03 nmol/l; P < 0.01), increased sensitivity of NKA to MBG, and an increased abundance of alpha1 NKA. Plasma levels of EO did not change, and the sensitivity of NKA to ouabain decreased. The transition to heart failure was accompanied by a decrease in alpha1 NKA, a reduction in plasma MBG, and decreased sensitivity of NKA to MBG. In addition, an increased abundance of ouabain-sensitive alpha3 NKA, a three-fold rise in plasma EO (1.01 +/- 0.13 versus 0.27 +/- 0.06 nmol/l), and a seven-fold increase in the ouabain sensitivity of NKA compared with controls were observed., Conclusions: During cardiac hypertrophy and the transition to heart failure, a shift in endogenous NKA ligands production is linked to a shift in myocardial NKA isoforms.
- Published
- 2004
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35. Myocardial PKC beta2 and the sensitivity of Na/K-ATPase to marinobufagenin are reduced by cicletanine in Dahl hypertension.
- Author
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Fedorova OV, Talan MI, Agalakova NI, Droy-Lefaix MT, Lakatta EG, and Bagrov AY
- Subjects
- Animals, Binding Sites, Blood Pressure drug effects, Bufanolides blood, Enzyme Inhibitors pharmacology, Hypertension metabolism, Hypertension physiopathology, Hypertrophy, Left Ventricular metabolism, Hypertrophy, Left Ventricular pathology, Hypertrophy, Left Ventricular physiopathology, Kidney metabolism, Kidney physiopathology, Protein Kinase C beta, Rats, Rats, Inbred Dahl, Sarcolemma enzymology, Sodium-Potassium-Exchanging ATPase chemistry, Sodium-Potassium-Exchanging ATPase metabolism, Antihypertensive Agents pharmacology, Bufanolides antagonists & inhibitors, Heart Ventricles enzymology, Hypertension enzymology, Protein Kinase C metabolism, Pyridines pharmacology, Sodium-Potassium-Exchanging ATPase antagonists & inhibitors
- Abstract
Marinobufagenin (MBG), an endogenous ligand of alpha-1 Na/K-ATPase, becomes elevated and contributes to hypertension in NaCl-loaded Dahl-S rats (DS). Protein kinase C (PKC) phosphorylates alpha-1 Na/K-ATPase and increases its MBG sensitivity. Cicletanine, an antihypertensive compound with PKC-inhibitory activity, reverses MBG-induced Na/K-ATPase inhibition and vasoconstriction. We hypothesized that increased PKC levels in sodium-loaded hypertensive DS would sensitize alpha-1 Na/K-ATPase to MBG and that PKC inhibition by cicletanine would produce an opposite effect. We studied the effects of cicletanine on systolic blood pressure, left ventricular PKC isoforms, cardiac alpha-1 Na/K-ATPase levels, and sensitivity to MBG in hypertensive DS. Seven DS received 50 mg x kg(-1) x d(-1) cicletanine, and 7 DS received vehicle during 4 weeks of an 8% NaCl diet. Vehicle-treated rats exhibited an increase in blood pressure, left ventricular mass, MBG excretion (74+/-11 vs 9+/-1 pmol/24 h, P<0.01), myocardial alpha-1 Na/K-ATPase protein, and PKC beta2 and delta. The sensitivity of Na/K-ATPase to MBG was enhanced at the level of high-affinity binding sites (IC50, 0.8 vs 4.4 nmol/L, P<0.01). Cicletanine-treated rats exhibited a 56-mm Hg reduction in blood pressure (P<0.01) and a 30% reduction in left ventricular weight, whereas cardiac alpha-1 Na/K-ATPase protein and MBG levels were unchanged. In cicletanine-treated rats, PKC beta2 was not increased, the sensitivity of Na/K-ATPase to MBG was decreased (IC50=20 micromol/L), and phorbol diacetate-induced alpha-1 Na/K-ATPase phosphorylation was reduced versus vehicle-treated rats. In vitro cicletanine treatment of sarcolemma from vehicle-treated rats also desensitized Na/K-ATPase to MBG, indicating that this effect was not solely attributable to a reduction in blood pressure. Thus, PKC-induced phosphorylation of cardiac alpha-1 Na/K-ATPase is a likely target for cicletanine treatment.
- Published
- 2003
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36. Effect of protein kinase C activation on Na+-H+ exchange in erythrocytes of frog Rana temporaria.
- Author
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Agalakova NI and Gusev GP
- Subjects
- Amiloride pharmacology, Animals, Anti-Arrhythmia Agents pharmacology, Biological Transport drug effects, Biological Transport physiology, Carcinogens pharmacology, Enzyme Activation physiology, Enzyme Inhibitors pharmacology, Phosphoprotein Phosphatases antagonists & inhibitors, Protein Kinase C antagonists & inhibitors, Rana temporaria, Sodium metabolism, Sodium Fluoride pharmacology, Sodium Radioisotopes, Staurosporine pharmacology, Tetradecanoylphorbol Acetate pharmacology, Amiloride analogs & derivatives, Erythrocytes enzymology, Protein Kinase C metabolism, Sodium-Hydrogen Exchangers metabolism
- Abstract
The treatment of frog erythrocytes incubated in standard nitrate medium with 100 nM phorbol ester (PMA) induced a sharp increase in the 22Na uptake by the cells and intracellular Na(+) concentration. The PMA-induced enhancement in 22Na uptake was stimulated by the addition of 0.1 mM ouabain to the incubation medium and completely blocked by 1 mM amiloride. The time course of 22Na uptake by frog red cells in the presence of PMA showed a lag phase ( approximately 5 min), after which was linear within 5-15 min. The calculated Na(+) influx in erythrocytes treated with PMA was 49.4+/-3.7 mmol l(-1) cells h(-1) as compared with 1.2+/-0.25 mmol l(-1) h(-1) for control cells. 5-(N-ethyl-N-isopropyl)-amiloride, selective blocker of NHE1, caused a dose-dependent inhibition of the PMA-induced Na(+) influx with IC(50) of 0.27 microM. The PMA-induced Na(+) influx was almost completely inhibited by 0.1 microM staurosporine, protein kinase C blocker. Pretreatment of frog red blood cells for 5, 10 or 15 min with 10 mM NaF, non-selective inhibitor of protein phosphatase, led to a progressive stimulation of the PMA effect on Na(+) influx. Both amiloride and NaF did not affect the basal Na(+) influx in frog erythrocytes. The data indicate that the Na(+)-H(+) exchanger in the frog erythrocytes is quiescent under basal conditions and can be markedly stimulated by PMA.
- Published
- 2003
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37. Marinobufagenin (MBG) suppression of ethanol-seeking behavior is associated with inhibition of brain cortex Na/K-ATPase in mice.
- Author
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Kashkin VA, Bagrov AY, Fedorova OV, Bagrov YY, Agalakova NI, Patkina NA, and Zvartau EE
- Subjects
- Alcohol Drinking metabolism, Animals, Behavior, Addictive enzymology, Bufanolides pharmacology, Cardenolides, Cerebral Cortex drug effects, Dose-Response Relationship, Drug, Enzyme Inhibitors pharmacology, Male, Mice, Mice, Inbred DBA, Saponins blood, Saponins metabolism, Saponins pharmacology, Sodium-Potassium-Exchanging ATPase metabolism, Alcohol Drinking drug therapy, Behavior, Addictive drug therapy, Bufanolides therapeutic use, Cerebral Cortex enzymology, Digoxin, Enzyme Inhibitors therapeutic use, Sodium-Potassium-Exchanging ATPase antagonists & inhibitors
- Abstract
In the present study the hypothesis was tested that sodium pump ligands (SPL) can modulate alcohol-seeking behavior and that this effect is related to changes in Na/K-ATPase activity in the central nervous system. Mice were tested for initiation of ethanol intravenous self-administration (IVSA) following i.p. pretreatment with vehicle or the endogenous SPL, marinobufagenin (MBG). Drug- and experimentally-naive mice acquired IVSA of 2% ethanol during a single 30-min session. MBG was found to dose-dependently attenuate (1.25-2.5 microg/kg) initiation of ethanol IVSA producing a decrease in the ratio and in the difference between operant responses of response-dependent and yoked animals as well as a decrease in percentage of mice demonstrating ethanol-seeking behavior. Attenuation of the reinforcing effect of ethanol resulting from MBG was associated with brain levels of this steroid capable of concurrently inhibiting Na/K-ATPase in the brain cortex. We hypothesize that endogenous digitalis-like factors could modulate the reinforcing effect of ethanol.
- Published
- 2002
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38. Marinobufagenin, an endogenous ligand of alpha-1 sodium pump, is a marker of congestive heart failure severity.
- Author
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Fridman AI, Matveev SA, Agalakova NI, Fedorova OV, Lakatta EG, and Bagrov AY
- Subjects
- Atrial Natriuretic Factor blood, Biomarkers blood, Blood Volume, Brachial Artery physiopathology, Cardenolides, Digoxin blood, Humans, Isoenzymes metabolism, Ligands, Male, Middle Aged, Saponins blood, Severity of Illness Index, Sodium-Potassium-Exchanging ATPase metabolism, Stroke Volume, Systole, Ventricular Function, Left, Bufanolides blood, Enzyme Inhibitors blood, Heart Failure blood, Heart Failure physiopathology, Vasoconstrictor Agents blood
- Abstract
Background: A reduced cardiac output in chronic heart failure (CHF) evokes renal NaCl and water retention, and, therefore, activates mechanisms promoting natriuresis. Atrial natriuretic peptide (ANP) is one such factor. We hypothesized that another NaCl sensitive endogenous natriuretic factor, i.e., marinobufagenin (MBG), a specific ligand of the alpha-1 subunit of Na/K ATPase (the main kidney isoform) and also a vasoconstrictor and cardiotonic substance, would be elevated in CHF patients in a graded manner with the severity of CHF., Methods and Results: We measured the plasma levels of MBG, alpha-hANP, ouabain-like compound (OLC) and left ventricular (LV) volumes and ejection fraction in 23 consecutive hypertensive male patients with CHF. Plasma MBG levels exhibited progressive increases (0.59 +/- 0.15, 1.08 +/- 0.20, 1.35 +/- 0.17 and 1.88 +/- 0.05 nmol/l NYHA 1-4, respectively) and paralleled the changes of alpha-hANP. Conversely, plasma OLC did not exhibit such increases. Plasma MBG correlated with alpha-hANP (r = 0.82; P < 0.0001). Both MBG and alpha-hANP correlated with LV systolic (r = 0.55 and r = 0.47; P < 0.01) diameter and inversely with ejection fraction (r = -0.73 and r = -0.60; P < 0.01). OLC did not exhibit correlations with alpha-hANP or LV volumes, but positively correlated with systolic brachial blood pressure and with pulse pressure., Conclusions: In CHF, MBG exhibits progressive increases similar to ANP, varies with CHF severity and correlates with LV systolic function. We hypothesize, that, in CHF, the concurrent production of these two natriuretic hormones, a vasorelaxant, ANP, and a vasoconstrictor, MBG, potentiate each other's natriuretic effects, but may offset their vasoactive actions.
- Published
- 2002
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39. Endogenous ligand of alpha(1) sodium pump, marinobufagenin, is a novel mediator of sodium chloride--dependent hypertension.
- Author
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Fedorova OV, Talan MI, Agalakova NI, Lakatta EG, and Bagrov AY
- Subjects
- Adrenal Glands metabolism, Animals, Antibodies pharmacology, Blood Pressure drug effects, Bufanolides antagonists & inhibitors, Cardenolides, Enzyme Inhibitors metabolism, Hypertension chemically induced, Ligands, Male, Models, Animal, Natriuresis drug effects, Pituitary Gland metabolism, Potassium blood, Rats, Rats, Inbred Dahl, Saponins antagonists & inhibitors, Saponins metabolism, Sodium blood, Sodium Chloride urine, Vasoconstrictor Agents metabolism, Bufanolides metabolism, Digoxin, Hypertension metabolism, Sodium Chloride, Dietary adverse effects, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
Background: Digitalis-like sodium pump ligands (SPLs) effect natriuresis via inhibition of renal tubular Na(+),K(+)-ATPase but may induce vasoconstriction. The present study investigated the potential roles of 2 putative endogenous SPLs, an ouabain-like compound (OLC) and an alpha(1) Na(+),K(+)-ATPase inhibitor, marinobufagenin (MBG), in regulating natriuresis and blood pressure (BP) responses to sustained and acute NaCl loading in Dahl salt-sensitive rats (DS)., Methods and Results: During 4 weeks of an 8% NaCl diet, DS exhibited a progressive increase in MBG renal excretion (66 +/-13 pmol/24 hours at week 4 versus 11 +/- 1 pmol/24 hours at baseline, n=48), which paralleled an increase in systolic BP (174 +/- 10 mm Hg at week 4 versus 110 +/- 2 mm Hg at baseline). By contrast, OLC excretion peaked at week 1 and returned to baseline levels. Administration of an anti-MBG, but not anti-ouabain antibody, to DS after 3 weeks of a high NaCl diet lowered BP (139 +/- 7 versus 175 +/- 5 mm Hg, P<0.001, n=5). Acute NaCl loading (2 hours) of DS (n=5) increased MBG and OLC excretion and natriuresis. Pretreatment of acutely NaCl-loaded DS with an anti-MBG antibody (n=5) reduced the excretion of sodium and MBG but not that of OLC. An anti-ouabain antibody (n=5) reduced sodium excretion and both OLC and MBG., Conclusions: An initial transient stimulation of OLC induced by NaCl loading of DS precedes an MBG response. A sustained increase in MBG production in DS contributes to the chronic BP elevation induced by a sustained high NaCl intake.
- Published
- 2002
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40. Marinobufagenin, an endogenous alpha-1 sodium pump ligand, in hypertensive Dahl salt-sensitive rats.
- Author
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Fedorova OV, Kolodkin NI, Agalakova NI, Lakatta EG, and Bagrov AY
- Subjects
- Animals, Blood Pressure drug effects, Bufanolides blood, Bufanolides isolation & purification, Bufanolides urine, Chromatography, High Pressure Liquid, Enzyme Inhibitors metabolism, Hypertension blood, Hypertension urine, Isoenzymes antagonists & inhibitors, Isoenzymes isolation & purification, Natriuresis, Ouabain blood, Ouabain urine, Rats, Rats, Inbred Dahl, Sodium blood, Sodium Chloride administration & dosage, Sodium, Dietary administration & dosage, Sodium-Potassium-Exchanging ATPase isolation & purification, Bufanolides metabolism, Hypertension metabolism, Kidney enzymology, Sodium-Potassium-Exchanging ATPase antagonists & inhibitors
- Abstract
Dahl salt-sensitive rats (DS), which have a mutation in the alpha-1 subunit of Na(+)/K(+)-ATPase, exhibit impaired pressure natriuresis and on a high-salt diet, retain Na(+) and exhibit increased blood pressure. Recently, we have shown that mammalian tissues contain a bufadienolide Na(+)/K(+)-ATPase inhibitory factor, marinobufagenin (MBG), that exhibits greater affinity for the alpha-1 than alpha-3 sodium pump isoform. The present study investigated the possible role of MBG in hypertension in DS on a high NaCl intake. Eight DS and 8 Dahl salt-resistant rats (DR) were placed on an 8% NaCl diet. Within 2 weeks, systolic blood pressure increased in DS (162+/-9 mm Hg at week 2 versus 110+/-2 mm Hg in baseline, P<0.01), and increased less in DR (124+/-3 mm Hg at week 2 versus 112+/-2 mm Hg in baseline). Renal excretion of MBG increased 4-fold (38.9+/-7.6 pmol versus 9.1+/-1.3 pmol in baseline, P<0.01) in DS, but by only 25% in DR (13.2+/-0.9 pmol versus 10.3+/-0.7 pmol in baseline). Excretion of endogenous ouabain did not change in either strain. MBG-immunoreactive material was purified from the urine of hypertensive DS by means of 2 steps of reverse-phase high performance liquid chromatography (HPLC) and compared with plant ouabain and amphibian MBG for its ability to inhibit the Na(+)/K(+)-ATPase from rat kidney (which expresses only alpha-1 Na(+)/K(+)-ATPase isoform). Unlike ouabain (IC(50)=248 micromol/L), serially diluted, HPLC-purified MBG immunoreactivity from DS and authentic MBG potently inhibited rat kidney Na(+)/K(+)-ATPase (IC(50)=70 and 78 nmol/L, respectively). Our results suggest that an alpha-1 Na(+)/K(+)-ATPase ligand, MBG, is elaborated to promote natriuresis in hypertensive DS. MBG acts as a selective inhibitor of the ouabain-resistant alpha-1 Na(+)/K(+)-ATPase subunit, ie, the major sodium pump isoform of the kidneys, as would be expected of a putative natriuretic hormone.
- Published
- 2001
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41. [Na, K-Pump activation by isoproterenol, methylxanthines, and iodoacetate in erythrocytes of the frog Rana temporaria].
- Author
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Gusev GP and Agalakova NI
- Subjects
- Aluminum Compounds pharmacology, Animals, Biological Transport, Active, Bucladesine pharmacology, Cholera Toxin pharmacology, Colforsin pharmacology, Cyclic AMP physiology, Cyclic GMP physiology, Enzyme Activation, Erythrocytes enzymology, Fluorides pharmacology, GTP-Binding Proteins physiology, In Vitro Techniques, Nitroprusside pharmacology, Potassium metabolism, Rana temporaria, Sodium metabolism, Erythrocytes drug effects, Iodoacetates pharmacology, Isoproterenol pharmacology, Sodium-Potassium-Exchanging ATPase metabolism, Xanthines pharmacology
- Published
- 2000
42. Effects of fluoride and vanadate on K+ transport across the erythrocyte membrane of Rana temporaria.
- Author
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Agalakova NI, Lapin AV, and Gusev GP
- Subjects
- Animals, Cell Membrane metabolism, Cell Membrane ultrastructure, Cold Temperature adverse effects, Erythrocytes metabolism, Erythrocytes ultrastructure, Hypotonic Solutions pharmacology, Isotonic Solutions pharmacology, Ouabain pharmacology, Potassium metabolism, Rana temporaria anatomy & histology, Rubidium Radioisotopes, Sodium-Potassium-Exchanging ATPase metabolism, Cell Membrane drug effects, Erythrocytes drug effects, Fluorides pharmacology, Rana temporaria metabolism, Sodium-Potassium-Exchanging ATPase drug effects, Vanadates pharmacology
- Abstract
K-Cl cotransport activity in frog erythrocytes was estimated as a Cl- -dependent component of K+ efflux from cells incubated in Cl- - or NO3- -containing medium at 20 degrees C. Decreasing the osmolality of the medium resulted in an increase in K+ efflux from the cells in a Cl- medium but not in an NO3- medium. Treatment of red cells with 5 mM NaF caused a significant decrease (approximately 50%) in K+ loss from the cells in iso- and hypotonic Cl- media but only a small decrease in K+ loss in isotonic NO3- medium. Addition of 1 mM vanadate to an isotonic Cl- medium also led to a significant reduction in K+ efflux. Similar inhibitory effects of NaF and vanadate on K+ efflux in a Cl- medium, but not in an NO3- medium were observed when the incubation temperature was decreased from 20 to 5 degrees C. Thus, under various experimental conditions, NaF and vanadate inhibited about 50% of Cl- -dependent K+ efflux from frog red cells probably due to inhibition of protein phosphatases. Cl- -dependent K+ (86Rb) influx into frog erythrocytes was nearly completely blocked (approximately 94%) by 5 mM NaF. In a NO3- medium, K+ influx was mainly mediated by the Na+,K+ pump and was unchanged in the presence of 5 mM NaF, 0.03 mM Al3+ or their combination. These data indicate that G proteins or cAMP are not involved in the regulation of Na+,K+ pump activity which is activated by catecholamines and phosphodiesterase blockers in these cells.
- Published
- 2000
43. Kinetics of K-Cl cotransport in frog erythrocyte membrane: effect of external sodium.
- Author
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Gusev GP, Agalakova NI, and Lapin AV
- Subjects
- Animals, Erythrocyte Membrane drug effects, Kinetics, Nystatin pharmacology, Ouabain pharmacology, Rana temporaria, Rubidium metabolism, Sodium metabolism, K Cl- Cotransporters, Carrier Proteins metabolism, Chlorides metabolism, Erythrocyte Membrane metabolism, Potassium metabolism, Symporters
- Abstract
In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO(3)) has been shown to mediate a large fraction of the total K(+) transport. In the present study, Cl(-)-dependent and Cl(-)-independent K(+) fluxes via frog erythrocyte membranes were investigated as a function of external and internal K(+) ([K(+)](e) and [K(+)](i)) concentration. The dependence of ouabain-resistant Cl(-)-dependent K(+) ((86)Rb) influx on [K(+)](e) over the range 0-20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K(m)) of 8.2 +/- 1.3 mm and maximal velocity (V(max)) of 10.4 +/- 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl(-)-dependent K(+) influx increased both K(m) (12.8 +/- 1.7 mm, P < 0.05) and V(max) (20.2 +/- 2.9 mmol/l/hr, P < 0.001). Raising [K(+)](e) above 20 mm in isotonic media significantly reduced the Cl(-)-dependent K(+) influx due to a reciprocal decrease of the external Na(+) ([Na(+)](e)) concentration below 50 mm. Replacing [Na(+)](e) by NMDG(+) markedly decreased V(max) (3.2 +/- 0.7 mmol/l/hr, P < 0.001) and increased K(m) (15.7 +/- 2.1 mm, P < 0.03) of Cl(-)-dependent K(+) influx. Moreover, NMDG(+) Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb(+) uptake and K(+) loss from red cells. Cell swelling did not affect the Na(+)-dependent changes in Rb(+) uptake and K(+) loss. In a nominally K(+)(Rb(+))-free medium, net K(+) loss was reduced after lowering [Na(+)](e) below 50 mm. These results indicate that over 50 mm [Na(+)](e) is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K(+), Cl(-)-dependent K(+) loss in K(+)-free media was a linear function of [K(+)](i), with a rate constant of 0.11 +/- 0.01 and 0.18 +/- 0.008 hr(-1) (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry with respect to transported K(+) ions. The residual, ouabain-resistant K(+) fluxes in NO(3) were only 5-10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K(+) effluxes in isotonic ( approximately 0. 014 hr(-1)) and hypotonic ( approximately 0.022 hr(-1)) media, but cell swelling resulted in a significant increase in the rate constants.
- Published
- 1999
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44. Inhibition and stimulation of K+ transport across the frog erythrocyte membrane by furosemide, DIOA, DIDS and quinine.
- Author
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Gusev GP, Lapin AV, and Agalakova NI
- Subjects
- Animals, Biological Transport drug effects, Chlorides blood, Erythrocyte Membrane drug effects, Kinetics, Ouabain pharmacology, Rana temporaria, Rubidium pharmacokinetics, 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid pharmacology, Carboxylic Acids pharmacology, Erythrocyte Membrane physiology, Furosemide pharmacology, Indenes pharmacology, Potassium blood, Quinine pharmacology
- Abstract
Frog erythrocytes were incubated in iso- or hypotonic media containing 10 mmol/l Rb+ and 0.1 mmol/l ouabain and both Rb+ uptake and K+ loss were measured simultaneously. Rb+ uptake by frog red cells in iso- and hypotonic media was reduced by 30-60% in the presence of 0.01-0.1 mmol/l [(dihydroindenyl)oxy] alkanoic acid (DIOA) or 0.5-1.0 mmol/l furosemide. Furosemide inhibited K+ loss from frog erythrocytes incubated in hypotonic media but did not affect it in isotonic media. DIOA at a concentration of 0.05 mmol/l inhibited of K+ loss from frog erythrocytes in both iso- and hypotonic media. At the concentrations of 0.01 and 0.02 mmol/l DIOA significantly suppressed K+ loss in a K+-free chloride medium but not in a K+-free nitrate medium. The Cl(-)-dependent K+ loss was completely blocked at a concentration of 0.1 mmol/l DIOA and the concentration required for 50% inhibition of K-Cl cotransport was approximately 0.015 mmol/l. However, the inhibitory effect of DIOA on K-Cl cotransport was masked by an opposite stimulatory effect on K+ transport which was also observed in nitrate medium. Quinine in a concentration of 0.2-1.0 mmol/l was able to inhibit Rb+ uptake and K+ loss only in hypotonic media. In isotonic media, quinine produced a stimulation of Rb+ uptake and K+ loss. A three to five-fold activation of Rb+ uptake and K+ loss was consistently observed in frog erythrocytes treated with 0.05-0.2 mmol/l 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). In contrast, another stilbene derivative 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) had no effect on K+ transport in the cells. Thus, of these drugs tested in the present study only DIOA at low concentrations may be considered as a selective blocker of the K-Cl cotransporter in the frog red blood cells.
- Published
- 1999
45. Differential effects of glycolytic and oxidative metabolism blockers on the Na-K pump in erythrocytes of the frog, Rana temporaria.
- Author
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Agalakova NI, Lapin AV, and Gusev GP
- Subjects
- Animals, Carbonyl Cyanide m-Chlorophenyl Hydrazone pharmacology, Cyanides pharmacology, Erythrocytes drug effects, In Vitro Techniques, Iodoacetates pharmacology, Iodoacetic Acid, Kinetics, Oxidative Phosphorylation drug effects, Potassium blood, Rana temporaria, Rotenone pharmacology, Rubidium blood, Uncoupling Agents pharmacology, Adenosine pharmacology, Deoxyglucose pharmacology, Erythrocytes metabolism, Glycolysis drug effects, Sodium-Potassium-Exchanging ATPase blood
- Abstract
This study was undertaken to evaluate the effects of various metabolic blockers on the Na-K-pump activity and ATP content of frog erythrocytes. To eliminate K-Cl cotransport, the frog erythrocytes were incubated in nitrate media at 20 degrees C. Incubation of the red cells in a glucose-free medium for 2 h had no effect on cell ATP content and K+ influx measured as 86Rb uptake for 60 min. The Na(+)-K(+)-pump activity was also unchanged in the frog erythrocytes incubated in a glucose-free medium containing 10 mM 2-deoxy-D-glucose or adenosine. Unexpectedly, the treatment of red cells with 1-2 mM glycolytic blocker iodoacetate produced a 2-fold increase in the ouabain-sensitive K+ influx. The cell ATP content declined by 9.4% after 2 h of cell incubation with iodoacetate. Incubation of the red cells for 90 min in the presence of 2 mM cyanide, 0.01 mM antimycin A or 5 mM azide resulted in a significant reduction in K+ influx by about 50%, 45% and 32%, respectively. The cell ATP content diminished over 60 min and 120 min of cell incubation with 2 mM cyanide by 15.6% and 31.7% of control levels, respectively. In time-course experiments, a 50% reduction in the K+ influx was observed when the frog erythrocytes were incubated for only 30 min in the presence of 2 mM cyanide. In contrast, 0.01-0.10 mM rotenone, a site I inhibitor, and 0.01 mM carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation were without effect on K+ influx into frog erythrocytes. These results indicate that about one-half of the Na(+)-K(+)-pump activity in frog erythrocytes is tightly functionally coupled to cytochromes via a separate "membrane-associated" ATP pool.
- Published
- 1997
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46. Temperature effects on ion transport across the erythrocyte membrane of the frog Rana temporaria.
- Author
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Agalakova NI, Lapin AV, and Gusev GP
- Subjects
- Animals, Bumetanide pharmacology, Carrier Proteins blood, Chlorides blood, Erythrocyte Membrane drug effects, Furosemide pharmacology, In Vitro Techniques, Ion Transport drug effects, Kinetics, Ouabain pharmacology, Potassium blood, Sodium blood, Temperature, K Cl- Cotransporters, Erythrocyte Membrane metabolism, Rana temporaria blood, Symporters
- Abstract
Unidirectional K+ and Na+ influxes in the frog erythrocytes incubated in Cl- or NO(3)- media with 2.7 mM K+ were measured using 86Rb and 22Na as tracers. K+ influx was inhibited by 35-55% in the presence of 0.2-1.0 mM furosemide but it was unaffected by 0.1-0.2 mM bumetanide. Furosemide at a concentration of 0.5 mM had no effect on K+ loss from the frog red cells incubated in a nominally K(+)-free medium. Together with our previous studies the data support the existence of K-Cl cotransport and the absence of Na-K-2Cl cotransport in the frog erythrocyte membrane. Cell cooling from 20 to 5 degrees C caused a decrease in K+ influx and K+ efflux via the K-Cl cotransporter (3.2- and 3.7-fold, respectively) giving an apparent energy of activation (EA) of about 60 kJ/mol and Q10 value of 2.5. Only small decline (approximately 30%) in the ouabain-sensitive K+ influx was found as temperature was changed from 20 to 5-10 degrees C. Low values of Q10 (approximately 1.5) and EA (27.3 kJ/mol) were obtained for passive K+ influx in the frog erythrocytes (ouabain-insensitive in NO(3)- medium) at temperature within 5-20 degrees C. However, the temperature coefficients were greater for passive Na+ influx and passive K+ efflux (Q10 approximately 2.4-2.5 and EA approximately 56-58 kJ/mol). The temperature dependence of all ion transport components displayed discontinuities showing no changes at temperature between 5 and 10 degrees C. Thus, cooling of the frog red cells is associated with a greater decrease of Na+ influx and K+ efflux than passive and active K+ influx. These data indicate that the preservation of a relative high activity of the Na,K-pump during cell cooling and also the temperature-induced changes in the K-Cl cotransport activity and ion passive diffusion contribute to maintenance of ion concentration gradients in the frog erythrocytes at decreased temperature.
- Published
- 1997
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47. Activation of the Na(+)-K+ pump in frog erythrocytes by catecholamines and phosphodiesterase blockers.
- Author
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Gusev GP, Agalakova NI, and Lapin AV
- Subjects
- 1-Methyl-3-isobutylxanthine pharmacology, Adrenergic Antagonists pharmacology, Animals, Biological Transport, Active drug effects, Enzyme Activation drug effects, Erythrocytes metabolism, In Vitro Techniques, Isoproterenol pharmacology, Kinetics, Ouabain pharmacology, Phentolamine pharmacology, Potassium blood, Propranolol pharmacology, Rana temporaria, Sodium blood, Theophylline pharmacology, Erythrocytes drug effects, Erythrocytes enzymology, Norepinephrine pharmacology, Phosphodiesterase Inhibitors pharmacology, Sodium-Potassium-Exchanging ATPase blood
- Abstract
K+ and Na+ influx into frog erythrocytes incubated in standard saline was studied using 86Rb and 22Na as tracers. 10 microM isoproterenol (ISP) produced a significant increase in K+ influx for the first 15 min, which was sustained during the entire 60 min of cell incubation. Treatment of red cells with the phosphodiesterase (PDE) blockers theophylline (THEO, 1 and 5 mM) or 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM) was also accompanied by an enhancement in K+ influx. A distinct additive effect on K+ influx into red cells was found when ISP and THEO or IBMX were added together. The increase in K+ transport induced by ISP plus IBMX was totally abolished by pretreatment of red cells with 0.1 mM ouabain. The ouabain-sensitive K+ influx in frog erythrocytes was elevated in the presence of ISP plus IBMX to 2.05 +/- 0.45, as compared with the control level of 0.39 +/- 0.11 mmol/L cells/hr (P < 0.001). Similar effects of ISP and IBMX on K+ influx were observed after chloride was replaced by nitrate. A dose-related increase in K+ influx into frog erythrocytes was observed at ISP concentrations of 10(-8)-10(-6) M, with a half-maximal stimulatory concentration of approximately 0.02 microM. The effects of ISP (10(-8)-10(-5) M) on K+ transport were completely abolished with 10 microM of the beta-adrenergic blocker propranolol, but alpha-adrenergic antagonists (phentolamine, prazosin, and yohimbine) did not alter the ISP-induced increase in K+ influx. The drugs tested had no effect on 22Na influx in frog red cells, but ISP produced a small decline (13%) in intracellular Na+ concentration. Thus, our study indicates that catecholamines and PDE blockers enhance K+ (86Rb) transport in frog erythrocytes mediated by Na(+)-K+ pump activity. The frog erythrocyte membrane may serve as a convenient model to investigate the hormonal modulation of the Na(+)-K+.
- Published
- 1996
- Full Text
- View/download PDF
48. [Potassium ion transport in the erythrocytes of the frog Rana ridibunda].
- Author
-
Agalakova NI, Lapin AV, and Gusev GP
- Subjects
- Animals, Biological Transport drug effects, Dose-Response Relationship, Drug, Erythrocyte Membrane drug effects, Erythrocyte Membrane metabolism, Erythrocytes drug effects, Furosemide pharmacology, Ouabain pharmacology, Potassium antagonists & inhibitors, Sodium blood, Sodium-Potassium-Exchanging ATPase blood, Sodium-Potassium-Exchanging ATPase drug effects, Erythrocytes metabolism, Potassium blood, Rana ridibunda blood
- Abstract
The K+ transport in isolated erythrocytes from the frog Rana ridibunda has been studied using 86Rb as a tracer at the temperature of 18-20 degrees C. At physiological K+ concentration (3 mM) in C1- medium ouabain and furosemide inhibited K+ influx by approximately 42 and 47%, respectively. Furosemide had no effect on the Na+ (22Na) transport under the same conditions. The replacement of C1- by NO-3 in medium resulted in significant decrease of total K+ influx, which was not inhibited by furosemide. The ouabain- and furosemide-sensitive components of K+ influx hyperbolically increased as a function of external K+e concentration (1-90 mM) in C1- medium and calculated values of Vmax were 2.2 +/- 0.14 and 5.8 +/- 1.2 mmol/1 cells/h, respectively. K+ influx into frog erythrocytes in the presence of both ouabain and furosemide in NO-3 medium was significantly lower compared with C1- medium. C1--dependent, furosemide-insensitive component of K+ influx increased in a saturable fashion in the range of K+e concentration from 1 to 90 mM. Residual component of K+ transport in NO3 medium in the presence of the blockers was a linear function of K+e concentration and is characterized by the constant of K+ influx rate equal to 0.028 +/- 0.002 h-1. The data obtained indicate that in parallel with the Na, K-pump specific K, C1-transporter participates in K+-transport in the frog erythrocyte membrane. The latter mechanism was only partially blocked by 1 mM furosemide.
- Published
- 1995
49. Potassium transport in red blood cells of frog Rana temporaria: demonstration of a K-Cl cotransport.
- Author
-
Gusev GP, Agalakova NI, and Lapin AV
- Subjects
- Animals, Chlorides pharmacology, Erythrocytes drug effects, Furosemide pharmacology, Nitrates pharmacology, Ouabain pharmacology, Potassium pharmacology, Sodium metabolism, Time Factors, K Cl- Cotransporters, Carrier Proteins drug effects, Erythrocytes metabolism, Potassium metabolism, Rana temporaria blood, Symporters
- Abstract
Pathways of K+ movement across the erythrocyte membrane of frog Rana temporaria were studied using 86Rb as a tracer. The K+ influx was significantly blocked by 0.1 mmol.l-1 ouabain (by 30%) and 1 mmol.l-1 furosemide (by 56%) in the red cells incubated in saline at physiological K+ concentration (2.7 mmol.l-1). Ouabain and furosemide had an additive effect on K+ transport in frog red cells. The ouabain-sensitive and furosemide-sensitive components of K+ influx saturated as f(K+)e with apparent Km values for external Ke+ concentration of 0.96 +/- 0.11 and 4.6 +/- 0.5 mmol.l-1 and Vmax of 0.89 +/- 0.04 and 2.8 +/- 0.4 mmol.l cells-1.h-1, respectively. The residual ouabain-furosemide-resistant component was also a saturable function of Ke+ medium concentration. Total K+ influx was significantly reduced when frog erythrocytes were incubated in NO3- medium. Furosemide did not affect K+ transport in frog red cells in NO3- media. At the same Ke+ concentration the ouabain-furosemide-insensitive K+ influx in Cl- medium was significantly greater than that in NO3- medium. We found no inhibitory effect of 1 mmol.l-1 furosemide on Na+ influx in frog red cells in Cl- medium. K+ loss from the frog erythrocytes in a K(+)-free medium was significantly reduced (mean 58%) after replacement of Cl- with NO3-. Furosemide (0.5 mmol.l-1) did not produce any significant reduction in the K+ loss in both media. The Cl(-)-dependent component of K+ loss from frog red cells was 5.7 +/- 1.2 mmol.l-1.h-1.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1995
- Full Text
- View/download PDF
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