The suggested involvement of ouabain in hypertension raised the need for a better understanding of its cellular action, but the mechanisms of ouabain toxicity are only now being uncovered. In the present study, we show that reduced glutathione (GSH) protected ouabain-sensitive (OS) cells from ouabain-induced toxicity and that the inhibition of GSH synthesis by D, L-buthionine-(S,R)-sulfoximine (BSO) sensitized ouabain-resistant (OR) cells. We could not observe formation of *OH or H2O2, but there was an increase in O2*-only in OS cells. Unexpectedly, an increased number of OR cells depolarized after treatment with ouabain, and BSO blocked this depolarization. Moreover, GSH increased ouabain-induced depolarization in OS cells. A sustained increase in tyrosine phosphorylation (P-Tyr) and Ras expression was observed after treatment of OS cells, and GSH prevented it. Conversely, BSO induced P-Tyr and Ras expression in ouabain-treated OR cells. The results obtained have three major implications: There is no direct correlation between membrane depolarization and ouabain-induced cell death; ouabain toxicity is not directly related to its classical action as a Na+, K+-ATPase inhibitor but seems to be associated to signal transduction, and GSH plays a major role in preventing ouabain-induced cell death.
Background and Aims: The steroid ouabain is found in plasma and in many mammalian tissues, and is now considered as a hormone. In the immune system, ouabain regulates a number of lymphocyte functions, but little is known about its effects on monocyte function. Monocytes are important for adequate immune responses. The aim of this work was to analyze the effect of ouabain on mCD14 expression, a surface molecule involved in the response against Gram-negative bacteria and phagocytosis. Methods: Human peripheral blood mononuclear cells obtained from healthy donors were separated by density gradient centrifugation. Monocytes were separated by adherence and treated for 24 h with 100 nM ouabain. mCD14, CD1a and P-p38 expression was analyzed by flow cytometry. Inhibitors of cell-signaling pathways, i.e. SB202190, reduced glutathione, rottlerin, tyrphostin A23, genistein, chelerythrine chloride, PD98059, PP1 and Ly 294002, were used concomitantly with ouabain to observe their effect on mCD14 expression. Results: Ouabain induced a significant decrease in mCD14 expression. This feature was not related to receptor endocytosis or cell death. Furthermore, mCD14 downregulation did not reflect a shift in differentiation into dendritic cells because this hormone failed to induce CD1a expression. Amongst several inhibitors of cell-signaling pathways triggered by ouabain, only epidermal growth factor receptor (EGFR) and p38 mitogen-activated protein kinase (MAPK) inhibitors (tyrphostin A23 and SB202109) significantly reverted the effect of ouabain on mCD14 expression. Accordingly, the levels of P-p38 were increased on monocytes after ouabain treatment. However, incubation with epidermal growth factor did not alter mCD14 expression. Conclusion: These findings suggest that ouabain downregulates mCD14 expression on monocytes through EGFR transactivation and p38 MAPK activation.
Vanadium is an environmentally toxic metal with peculiar and sometimes contradictory cellular effects. It is insulin-mimetic, it can either stimulate cell growth or induce cell death, and it has both mutagenic and antineoplastic properties. However, the mechanisms involved in those effects are poorly understood. Several studies suggest that H(2)O(2) is involved in vanadate-induced cell death, but it is not known whether cellular sensitivity to vanadate is indeed related to H(2)O(2) generation. In the present study, the sensitivity of four cell lines from different origins (K562, K562-Lucena 1, MDCK, and Ma104) to vanadate and H(2)O(2) was evaluated and the production of H(2)O(2) by vanadate was analyzed by flow cytometry. We show that cell lines very resistant to H(2)O(2) (K562, K562-Lucena 1, and Ma104 cells) are much more sensitive to vanadate than MDCK, a cell line relatively susceptible to H(2)O(2), suggesting that vanadate-induced cytotoxicity is not directly related to H(2)O(2) responsiveness. In accordance, vanadate concentrations that reduced cellular viability to approximately 60-70% of the control (10 mumol/L) did not induce H(2)O(2) formation. A second hypothesis, that peroxovanadium (PV) compounds, produced once vanadate enters into the cells, are responsible for the cytotoxicity, was only partially confirmed because MDCK cells were resistant to both vanadate and PV compounds (10 micromol/L each). Therefore, our results suggest that vanadate toxicity occurs by two distinct pathways, one dependent on and one independent of H(2)O(2) production.
Chagas disease is a neglected disease caused by the protozoan Trypanosoma cruzi and affects 8 million people worldwide. The main chemotherapy is based on benznidazole. The efficacy in the treatment depends on factors such as the parasite strain, which may present different sensitivity to treatment. In this context, the expression of ABC transporters has been related to chemotherapy failure. ABC transporters share a well-conserved ABC domain, responsible for ATP binding and hydrolysis, whose the energy released is coupled to transport of molecules through membranes. The most known ABC transporters are ABCB1 and ABCC1, involved in the multidrug resistance phenotype in cancer, given their participation in cellular detoxification. In T. cruzi, 27 ABC genes were identified in the genome. Nonetheless, only four ABC genes were characterized: ABCA3, involved in vesicular trafficking; ABCG1, overexpressed in strains naturally resistant to benznidazole, and P-glycoprotein 1 and 2, whose participation in drug resistance is controversial. Considering P-glycoprotein genes are related to ABCC subfamily in T. cruzi according to the demonstration using BLASTP alignment, we evaluated both ABCB1-like and ABCC-like activities in epimastigote and trypomastigote forms of the Y strain. The transport activities were evaluated by the efflux of the fluorescent dyes Rhodamine 123 and Carboxyfluorescein in a flow cytometer. Results indicated that there was no ABCB1-like activity in both T. cruzi forms. Conversely, results demonstrated ABCC-like activity in both epimastigote and trypomastigote forms of T. cruzi. This activity was inhibited by ABCC transport modulators (probenecid, indomethacin, and MK-571), by ATP-depleting agents (sodium azide and iodoacetic acid) and by the thiol-depleting agent N-ethylmaleimide. Additionally, the presence of ABCC-like activity was supported by direct inhibition of the thiol-conjugated compound efflux with indomethacin, characteristic of ABCC subfamily members. Taken together, the results provide the first description of native ABCC-like activity in T. cruzi epimastigote and trypomastigote forms, indicating that the study of the biological role for that thiol transporter is crucial to reveal new molecular mechanisms for therapeutic approaches in the Chagas disease.
Although the kidney is a major target in hypertension, several studies have correlated important immune alterations with the development of hypertension in spontaneously hypertensive rats (SHR), like increased secretion of pro-inflammatory cytokines, inflammatory infiltration in kidneys and thymic atrophy. Because adenosine-triphosphate-binding cassette sub-family B member 1 (ABCB1; P-glycoprotein) and adenosine-triphosphate-binding cassette sub-family C member 1 (ABCC1; multidrug resistance protein 1), two proteins first described in multidrug resistant tumors, physiologically transport several immune mediators and are required for the adequate functioning of the immune system, we aimed to measure the expression and activity of these proteins in peripheral blood mononuclear cells (PBMC), thymocytes, and also kidneys of normotensive Wistar Kyoto rats and SHR. Our results showed that ABCB1, but not ABCC1, activity was diminished (nearly 50%) in PBMC. Moreover, Abcb1b gene was downregulated in PBMC and kidney of SHR and this was not counterbalanced by an upregulation of its homolog Abcb1a, suggesting that the diminished activity is due to downregulation of the gene. No alteration was detected in ABCB1 activity in SHR thymocytes, indicating that this downregulation occurs after lymphocytes leave the primary lymphoid organs. Even though it is not known at present which parameter(s) is(are) responsible for this downregulation, it may contribute for the altered immune response observed in hypertension and to possible altered drug disposition in hypertensive individuals, resulting in greater drug interaction and increased drug toxicity.
Besides being a (Na(+),K(+))-ATPase inhibitor, high doses of the hormone ouabain have also been reported to modulate both the expression and activity of proteins belonging to the ATP binding cassette family of transporters, such as ABCC7 (CFTR), ABCB1 (P-glycoprotein), and ABCC1 (MRP1). Although these proteins are present in the kidney, only ABCB1 has a putative physiological role in this organ, secreting endobiotics and xenobiotics. In the present work, we studied the relationship between ouabain and ABCC1 expression and function, aiming to establish a physiological role for ouabain. It was observed that prolonged (24 h) but not short (30 min) incubation with 1 nmol/L or higher ouabain concentrations decreased the expression of ABCC1 protein and induced its mRNA expression. This decrease was rapidly reversible, reaching control levels after incubation of cells in ouabain-free medium for 3 h, denoting a hormonal action. Moreover, concentrations equal or higher than 100 nmol/L ouabain also induced impairment of ABCC1 activity, increasing the accumulation of carboxyfluorescein diacetate, an ABCC1 fluorescent substrate. Because ouabain is now accepted as an endogenous hormone, our results suggest that ABCC1 is regulated by hormones related to body volume control, which may have implications for the treatment of hypertensive cancer patients. Moreover, providing ABCC1 is expressed in several other tissues, such as brain, testis, and the immune system, and is related to the transport of glutathione, it is possible that ouabain release may control a number of functions within these organs and tissues by modulating both the expression and the activity of ABCC1.
The biological effects of electromagnetic waves are widely studied, especially due to their harmful effects, such as radiation-induced cancer and to their application in diagnosis and therapy. However, the biological effects of sound, another physical agent to which we are frequently exposed have been considerably disregarded by the scientific community. Although a number of studies suggest that emotions evoked by music may be useful in medical care, alleviating stress and nociception in patients undergoing surgical procedures as well as in cancer and burned patients, little is known about the mechanisms by which these effects occur. It is generally accepted that the mechanosensory hair cells in the ear transduce the sound-induced mechanical vibrations into neural impulses, which are interpreted by the brain and evoke the emotional effects. In the last decade; however, several studies suggest that the response to music is even more complex. Moreover, recent evidence comes out that cell types other than auditory hair cells could response to audible sound. However, what is actually sensed by the hair cells, and possible by other cells in our organism, are physical differences in fluid pressure induced by the sound waves. Therefore, there is no reasonable impediment for any cell type of our body to respond to a pure sound or to music. Hence, the aim of the present study was to evaluate the response of a human breast cancer cell line, MCF7, to music. The results' obtained suggest that music can alter cellular morpho-functional parameters, such as cell size and granularity in cultured cells. Moreover, our results suggest for the 1 st time that music can directly interfere with hormone binding to their targets, suggesting that music or audible sounds could modulate physiological and pathophysiological processes.
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