Navegando por Palavras-chave "intracellular calcium"
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- ItemSomente MetadadadosActivation of P2Y(1) receptor triggers two calcium signaling pathways in bone marrow erythroblasts(Elsevier B.V., 2006-03-18) Paredes-Gamero, E. J.; Craveiro, R. B.; Pesquero, J. B.; Franca, J. P.; Oshiro, MEM; Ferreira, A. T.; Universidade Federal de São Paulo (UNIFESP)In this study, we describe the presence of P2 receptor subtypes and Ca2+ signaling in erythroblasts. ATP and ADP produced a biphasic increase of intracellular Ca2+ concentration ([Ca2+],), with an initial transient phase followed by a sustained phase. Reverse transcription polymerase chain reaction (RT-PCR) showed the expression of P2Y(1), P2Y(2) and P2Y(12). the selective P2Y(1) receptor antagonist 2'-deoxy-N-6-methyl-adenosine- 3',5'diphosphate (MRS2179) and the Gi protein inhibitor pertussis toxin blocked Ca2+ increase. the initial transient [Ca2+](i) increase phase was sensitive to the 1,4,5-inositol trisphosphate (IP3) receptor blocker 2-aminoethoxy-diphenylborate (2-APB), while the sustained phase was sensitive to the protein kinase C (PKC) inhibitor 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3(1H-indol-3-yl)-maleimide (GF109203X) and calcium calmodulin kinase 11 (CaMKII) inhibitor 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62). in addition, the PKC activator phorbol-12,13-dibutyrate (PDBu) produced increase of [Ca2+](i). Flow cytometry analysis showed the expression of Ca2+-dependent PKC alpha, beta I, gamma and phospho-CaMKII.These results suggest that the activation of the P2Y(1) receptor triggers two different [Ca2+]i increase pathways, one IP3-dependent and the other kinase-dependent. (c) 2006 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosCyclosporin A tubular effects contribute to nephrotoxicity: role for Ca2+ and Mg2+ ions(Oxford Univ Press, 2003-11-01) Costa, Maristela Carvalho da; Castro, Isac de; Cuvello Neto, Américo L; Ferreira, Alice T. [UNIFESP]; Burdmann, Emmanuel A.; Yu, Luis; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP); Sao Jose do Rio Preto Med SchBackground. Cyclosporin A (CsA) nephrotoxicity has been attributed primarily to renal haemodynamic alterations caused by afferent arteriolar vasoconstriction. However, CsA nephropathy is also characterized by CsA-induced pre-glomerular disturbances and interstitial injury that may occur independently of haemodynamic changes. Given the high lipophilic activity of CsA, we hypothesized that direct tubular injury is likely to contribute to nephrotoxicity.Methods. To investigate tubular toxicity of CsA, increasing concentrations of CsA (1, 2.5, 10, 25, 50 and 100 mug/ml) and its vehicle (cremophor) were added to isolated rat proximal tubules (PT). Cell injury was assessed by lactate dehydrogenase (LDH) release. the role of Ca2+ ions in tubular toxicity and the effect of calcium channel blockers on CsA toxicity were evaluated by measuring intracellular calcium using the fluorescent dye Fura-2 AM. the role of Mg2+ ions was assessed using high extracellular Mg2+ medium (2 in M).Results. Whereas cremophor alone was not toxic to PT, CsA caused PT injury but only at the highest concentration (100 mug/ml). After 90 min incubation, LDH was 22.5% in control PT and 41.9% in PT treated with 100 mug/ml CsA (P < 0.001, n = 11). There was a transient increase in intracellular calcium ([Ca2+]i) after CsA administration. A low calcium medium (100 nM) prevented CsA injury to renal tubules. However, verapamil, but not nifedipine, enhanced cell damage. Only nifedipine completely prevented [Ca 21]i increases following CsA. Finally, a high Mg2+ medium attenuated CsA-induced injury.Conclusion. We found that high CsA concentrations caused Ca2+- and Mg2+-dependent PT injury. Thus, low extracellular Ca2+ and high Mg2+ media attenuated CsA-induced tubular injury. Verapamil, but not nifedipine, enhanced CsA tubular toxicity. Therefore, CsA-induced tubular injury may contribute to CsA nephrotoxicity independently of haemodynamic disturbances.
- ItemSomente MetadadadosEffect of cyclosporin A on nitric oxide production in cultured LLC-PK1 cells(Marcel Dekker Inc, 2001-01-01) Lima, R.; Serone, A. P.; Schor, N.; Higa, EMS; Universidade Federal de São Paulo (UNIFESP)The effect of Cyclosporin A on nitric oxide production was studied in cultured LLC- PK1 cells. for this purpose the cells were incubated with vehicle (olive oil, 10 mug/ml in DMSO), Cyclosporin A (CsA, 10 mug/ml), tumor necrosis factor (TNF-alpha, 150 U/ml) + interferon (IFN-gamma, 500 U/ml) to upregulate NOS synthesis, and therefore, NO production (used as a positive control), or CsA + TNF-alpha + IFN-gamma. After 72 hours the culture medium was collected and nitrite was determined by the Griess method. the results were normalized to the protein harvested from these cells as measured by the Lowry method. Viability was determined by the exclusion of the fluorescent dyes (acridine orange and ethidium bromide). Intracellular calcium was measured spectrophotometrically using the fluorescent calcium indicator fura-2 AM. in CsA treated cells, the nitrite (pmoles/mg of protein) was decreased when compared to control (12.8 +/- 0.5 vs. 18.3 +/- 0.6; p < 0.05; both n = 8). TNF- + IFN-gamma increased the nitrite synthesis (52.0 +/- 0.2; p < 0.05 vs. control; n = 6). This effect was decreased significantly by the simultaneous treatment with CsA (38.8 +/- 0.3; p < 0.05; n = 6). Cell viability in CsA group was decreased when compared to the cdntrol (84.7 +/- 0.2% vs. 93.6 +/- 0,1%; p < 0.05: both n = 10). TNF- +/- IFN-gamma had no effect on viability (93.0 +/- 0.3%; n = 10). However, when combined with CsA, viability was decreased relative to the control (85.0 +/- 0.2%; p < 0.05; n = 10). Acute (1 h) or chronic (72 h) treatment of LLC- PK1 cells with CsA had no effect on basal calcium levels.Our results demonstrate a reduced level of nitric oxide production in LLC- PK1 cells treated with CsA. There was no effect of the drug on intracellular calcium levels, however CsA treatment did reduce cellular viability. We suggest that, in part, the decreased levels of NO production are a secondary consequence of direct cell damage. However, CsA may also be exerting direct effects on NO synthesis through its interactions with both iNOS and cNOS. These results also provide a dual mechanism of action for CsA induced nephrotoxicity, that is, direct cell damage and interference with the NO system within the nephron.
- ItemSomente MetadadadosEffects of arachnotoxin on intracellular pH and calcium in human spermatozoa(Elsevier B.V., 2007-06-01) Romero, Fernando; Cunha, Maria Adelaide [UNIFESP]; Sanchez, Raul; Ferreira, Alice Teixeira [UNIFESP]; Schor, Nestor [UNIFESP]; Oshiro, Maria Etsuko Miyamoto [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ La FronteraObjective: To determine the effect of arachnotoxin (ATx), a venom extracted from the Chilean spider Latrodectus mactans, on intracellular calcium ([Ca2+](i)) and pH (pH(i)) in capacitated human spermatozoa.Design: Spermatozoa were collected from fertile adult men (n = 8). Mobile spermatozoa were collected by the swim up technique and stimulated with the crude extract of ATx and with progesterone (P).Setting: Hospital of the Federal University of São Paulo, São Paulo, Brazil.Main outcome measure(s): [Ca2+](i) was measured in fura2-AM-loaded spermatozoa, and pHi was measured in spermatozoa loaded with the pH-sensitive dye [(2',7')-bis (carboxymethyl)-(5,6)-carboxyfluorescein]-AM (BCECF).Result(s): the ATx and P induced a biphasic change in [Ca2+](i) consisting of a peak followed by a small but sustained elevation. the response to ATx was greatly reduced by pretreatment with P. the ATx caused intracellular acidification, whereas P induced alkalinization. Blockade of the NA(+)/H+ exchanger with ethylisopropylamiloride (EIPA) sharply increased ATx-induced acidification.Conclusion(S): Arachnotoxin increased [Ca2+](i) through the opening of calcium channels and release of calcium from intracellular stores. the ATx reduced pHi in human sperm, possibly by inhibiting the Na+/H+ exchanger.
- ItemSomente MetadadadosEscherichia coli lipopolysaccharide impairs the calcium signaling pathway in mesangial cells: role of angiotensin II receptors(Soc Experimental Biology Medicine, 2010-06-01) Maquigussa, Edgar [UNIFESP]; Arnoni, Carine P. [UNIFESP]; Cristovam, Priscila C. [UNIFESP]; Oliveira, Andrea S. de [UNIFESP]; Higa, Elisa M. S. [UNIFESP]; Boim, Mirian A. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Sepsis causes impaired vascular reactivity, hypotension and acute renal failure. the ability of the Escherichia coli endotoxin (lipopolysaccharide [LPS]) to impair agonist-induced contractility in mesangial cells, which contributes to LPS-induced renal dysfunction, was evaluated. Agonist-induced intracellular calcium ([Ca(2+)]i) mobilization was analyzed using angiotensin II (AngII). the effect of LPS on the levels of the renin-angiotensin system (RAS) components and the roles of vasodilatation-inducing molecules including AT2 receptor (AT2R) and nitric oxide (NO) in the cell reactivity were also evaluated. Confluent human mesangial cells (HMCs) were stimulated with LPS (0111-B4, 100 mu g/mL). AngII-induced [Ca(2+)]i mobilization was measured by fluorometric analysis using Fura-2AM in the absence and presence of an AT2R antagonist (PD123319). the mRNA and protein levels for angiotensinogen, renin, angiotensin-converting enzyme, AT1R and AT2R were analyzed by realtime reverse transcriptase-polymerase chain reaction and Western blot, respectively. NO production was measured by the chemiluminescence method in the culture media after 24, 48 and 72 h of LPS incubation. After 24 h, LPS-stimulated HMCs displayed lower basal [Ca(2+)]i and an impaired response to AngII-induced rise in [Ca(2+)]i. LPS significantly increased AT2R levels, but did not cause significant alterations of RAS components. PD123319 restored both basal and AngII-induced [Ca(2+)]i peak, suggesting an involvement of AT2R in these responses. the expected increase in NO production was significant only after 72 h of LPS incubation and it was unaffected by PD123319. Results showed that LPS reduced the reactivity of HMCs to AngII and suggest that the vasodilatation induced by AT2R is a potential mediator of this response through a pathway independent of NO.
- ItemSomente MetadadadosThe Natural Cell-Penetrating Peptide Crotamine Targets Tumor Tissue in Vivo and Triggers a Lethal Calcium-Dependent Pathway in Cultured Cells(Amer Chemical Soc, 2012-02-01) Nascimento, Fabio D.; Sancey, Lucie; Pereira, Alexandre; Rome, Claire; Oliveira, Vitor [UNIFESP]; Oliveira, Eduardo B.; Nader, Helena B. [UNIFESP]; Yamane, Tetsuo; Kerkis, Irina; Tersariol, Ivarne L. S.; Coll, Jean-Luc; Hayashi, Mirian A. F. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Bandeirante São Paulo UNIBAN; Univ Grenoble 1; Inst Butantan; Universidade de São Paulo (USP); CBA; Univ Mogi das CruzesOur goal was to demonstrate the in vivo tumor specific accumulation of crotamine, a natural peptide from the venom of the South American rattlesnake Crotalus durissus terrificus, which has been characterized by our group as a cell penetrating peptide with a high specificity for actively proliferating cells and with a concentration-dependent cytotoxic effect. Crotamine cytotoxicity has been shown to be dependent on the disruption of lysosomes and subsequent activation of intracellular proteases. in this work, we show that the cytotoxic effect of crotamine also involves rapid intracellular calcium release and loss of mitochondrial membrane potential as observed in real time by confocal microscopy. the intracellular calcium overload induced by crotamine was almost completely blocked by thapsigargin. Microfluorimetry assays confirmed the importance of internal organelles, such as lysosomes and the endoplasmic reticulum, as contributors for the intracellular calcium increase, as well as the extracellular medium. Finally, we demonstrate here that crotamine injected intraperitoneally can efficiently target remote subcutaneous tumors engrafted in nude mice, as demonstrated by a noninvasive optical imaging procedure that permits in vivo real-time monitoring of crotamine uptake into tumor tissue. Taken together, our data indicate that the cytotoxic peptide crotamine can be used potentially for a dual purpose: to target and detect growing tumor tissues and to selectively trigger tumor cell death.
- ItemSomente MetadadadosSoluble uric acid increases intracellular calcium through an angiotensin II-dependent mechanism in immortalized human mesangial cells(Soc Experimental Biology Medicine, 2010-07-01) Albertoni, Guilherme [UNIFESP]; Maquigussa, Edgar [UNIFESP]; Pessoa, Edson [UNIFESP]; Barreto, Jose Augusto; Borges, Fernanda [UNIFESP]; Schor, Nestor [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Assoc Beneficente Coleta Sangue ColsanHyperuricemia is associated with increases in cardiovascular risk and renal disease. Mesangial cells regulate glomerular filtration rates through the release of hormones and vasoactive substances. This study evaluates the signaling pathway of uric acid (UA) in immortalized human mesangial cells (ihMCs). To evaluate cell proliferation, ihMCs were exposed to UA (6-10 mg/dL) for 24-144 h. in further experiments, ihMCs were treated with UA (6-10 mg/dL) for 12 and 24 h simultaneously with losartan (10(-7) mmol/L). Angiotensin II (All) and endothelin-1 (ET-1) were assessed using the enzyme-linked immunosorbent assay (ELISA) technique. Pre-pro-ET mRNA was evaluated by the real-time PCR technique. It was observed that soluble UA (8 and 10 mg/dL) stimulated cellular proliferation. UA (10 mg/dL) for 12 h significantly increased All protein synthesis and ET-1 expression and protein production was increased after 24 h. Furthermore, UA increased [Ca(2+)](i), and this effect was significantly blocked when ihMCs were preincubated with losartan. Our results suggested that UA triggers reactions including All and ET-1 production in mesangial cells. in addition, UA can potentially affect glomerular function due to UA-induced proliferation and contraction of mesangial cells. the latter mechanism could be related to the long-term effects of UA on renal function and chronic kidney disease.