Heparin prevents the cytotoxic activity of Bothrops jararacussu and Apis mellifera venoms in renal cells.

Envenomation by Bothrops snakes and Apis mellifera bee may imply systemic disorders which affect well-perfused organs such as kidneys, a process that can lead to acute renal failure. Nevertheless, there is scarce information regarding a direct renal cell effect and the putative antagonism by antivenoms. Here the cytotoxic effect of B. jararacussu and A. mellifera venoms was evaluated in the renal proximal tubule cell line LLC-PK1, as well as the antagonism of this effect by heparin. B. jararacussu venom showed significant cytotoxicity as assessed by LDH release and MTT reduction, with a sharp decline of the cell number after 180 min (>90% at 50 µg/mL). A. mellifera venom produced a much faster and potent cytotoxic activity, conferring almost no viable cells after 15 min at 25 µg/mL. Phase contrast microscopy revealed that while B. jararacussu venom induced a progressive loss of cell adhesion and detachment, A. mellifera venom promoted a rapid plasma membrane disruption and nuclear condensation suggestive of necrotic cell death. Pre-incubation of both venoms with heparin for 30 min significantly reduced cytotoxicity. Our results demonstrate direct toxicity of B. jararacussu and A. mellifera venoms toward renal cells but with distinct kinetics and cell pattern, suggesting different mechanisms of action. In addition, the antagonistic, cytoprotective effect of heparin ascribes such compound as a promising drug for preventing renal failure from envenomation.

Glucocorticoid susceptibility and in vivo ABCB1 activity differ in murine B cell subsets.

Glucocorticoids are produced and released by the adrenal gland and become elevated in response to stress. Although glucocorticoids are well known for their immunosuppressive effects, less is known about their effects on B cells. ABCB1 is an efflux pump expressed in both cancer and normal cells, modulating the gradient of various metabolites, including hydrocortisone. Our goal was to evaluate the effect of this glucocorticoid on murine B cell differentiation and whether sensitivity to hydrocortisone could be related to ABCB1 activity in vivo. C57BL/6 mice received one or three consecutive i.p. injections of hydrocortisone (70, 140 and 200 mg/kg/day). ABCB1 activity was evaluated via the rhodamine-123 transport and inhibited by cyclosporin A in hydrocortisone-treated and control mice. Cells from bone marrow, spleen and blood were counted, incubated with antibodies and analyzed by flow cytometry. A single hydrocortisone injection did not alter the number of bone marrow subsets. Conversely, three daily injections were able to reduce the cell number of most bone marrow subsets, excepting c-kit-sca-1+ and mature B cells. This treatment reduced marginal zone, follicular and transitional B cells, though splenic subsets were more resistant than bone marrow B cells. Recirculating follicular B cells in the blood were resistant to hydrocortisone. With the exception of follicular B cells, all subpopulations exhibited ABCB1 activity. However, hydrocortisone treatment did not affect ABCB1 activity in most subsets analyzed. Results suggest that hydrocortisone is able to regulate B cell lymphopoiesis although ABCB1 activity is not related to the susceptibility to that glucocorticoid in B cell subsets.

Functional Characterization of ABCC Proteins from Trypanosoma cruzi and Their Involvement with Thiol Transport.

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.

Direct effects of music in non-auditory cells in culture.

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.

Ouabain inhibits monocyte activation in vitro: prevention of the proinflammatory mCD14(+)/CD16(+) subset appearance and cell-size progression.

Classically described as a potent inhibitor of the sodium-potassium adenosine triphosphatase enzyme, ouabain has been further shown to act as an effective immunomodulator in mammals. Recently, our group showed that this hormone downregulates membrane CD14 (mCD14) in human monocytes, though it is not known whether monocyte activation status could modify ouabain influence. Hence, we aimed to investigate ouabain effect during monocyte activation in vitro, analyzing mCD14, CD16 and CD69 expression in total monocytes after two periods of adhesion (2 hours and 24 hours) or in small and large monocyte subpopulations separately. Ouabain (100 nM) inhibited monocyte-size increase, characteristic of activation, only when added to cells immediately after 2 hours' adhesion. Moreover, downregulation of both mCD14 and CD16 expression by ouabain was more effective in small monocytes and in cells after 2 hours' adhesion. Since monocytes after 24 hours' adhesion showed no lack of ouabain binding and no CD69 upregulation, it seems that ouabain is somehow incapable of triggering an appropriate cell-signaling induction once monocytes become activated. Furthermore, though p38 MAPK activation was crucial for the impairment in cell-size progression induced by ouabain, its inhibition did not alter ouabain-induced CD69 upregulation, suggesting that other molecules may participate in the response to this hormone by monocytes. Our data suggest that ouabain inhibits monocyte activation in vitro, preventing both cell-size increase and the appearance of the proinflammatory mCD14(+)/CD16(+) subpopulation. Thus, the findings suggest that individuals suffering from disorders commonly associated with high ouabain plasma levels, like hypertension, may present defective monocyte activation under inflammation or infection.

Diverse actions of ouabain and its aglycone ouabagenin in renal cells.

The cellular actions of ouabain are complex and involve different pathways, depending on the cell type and experimental conditions. Several studies have reported that Madin-Darby canine kidney (MDCK) cellular sensitivity to ouabain is not related to Na-K-ATPase inhibition, and others showed that some cell types, such as Ma104, are resistant to ouabain toxicity albeit their Na-K-ATPase isoforms possess high affinity for this glycoside. We describe here that the effects of ouabain and ouabagenin also diverge in MDCK and Ma104 cells, being MDCK cells more resistant to ouabagenin, while Ma104 cells are resistant to both molecules. This feature seems to correlate with induction of cell signaling, since ouabain, but not ouabagenin, induced an intense and sustained increase in tyrosine phosphorylation levels in MDCK cells. Moreover, ouabain-induced phosphorylation in Ma104 cells was approximately half than that observed in MDCK cells. The proportion between alpha and beta subunits of Na-K-ATPase was similar in MDCK cells, though Ma104 cells presented more alpha subunits, located mainly at the cytoplasm. Furthermore, a fluorescent ouabain-analog labeled mainly the cytoplasm of Ma104 cells, the opposite of that seen in MDCK cells, corroborating the results using anti-Na-K-ATPase antibodies. Hence, the results suggest that ouabain and ouabagenin differ in terms of Na-K-ATPase inhibition and cell signaling activation in MDCK cells. Additionally, MDCK and Ma104 cell lines respond differently to ouabain, perhaps due to an intrinsic ability of this glycoside to selectively reach the cytoplasm of Ma104 cells.

ABCB1 (P-glycoprotein) but not ABCC1 (MRP1) is downregulated in peripheral blood mononuclear cells of spontaneously hypertensive rats.

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.

Mechanisms of ouabain toxicity.

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.