RESUMO
INTRODUCTION: Hydrochlorothiazide has a negative influence on penile erection but little is known about the mechanism(s) involved. AIMS: To characterize the effects of this diuretic on mouse corpus cavernosum (CC) smooth muscle in vitro and ex vivo. METHODS: CC strips of C57BL/6 mice (12-16 weeks old) were mounted in organ baths containing Krebs-Henseleit solution and tissue reactivity was evaluated. Expression of genes encoding diuretic targets and enzymes involved in penile erection were evaluated by polymerase chain reaction. MAIN OUTCOME MEASURES: Stimulation-response curves to phenylephrine (10 nmol/L-100 µmol/L) or to electrical field stimulation (1-32 Hz) were constructed, with or without hydrochlorothiazide. Strips of CC from mice after long-term hydrochlorothiazide treatment (6 mg/kg/day for 4 weeks) with or without amiloride (0.6 mg/kg/day for 4 weeks) in vivo also were studied. Nitric oxide and Rho-kinase pathways were evaluated. RESULTS: Hydrochlorothiazide (100 µmol/L) increased the maximum response to phenylephrine by 64% in vitro. This effect was unaffected by the addition of indomethacin (5 µmol/L) but was abolished by N((ω))-nitro-L-arginine methyl ester (100 µmol/L). Hydrochlorothiazide (100 µmol/L) potentiated electrical field stimulation-induced contraction in vitro, but not ex vivo. Long-term treatment with hydrochlorothiazide increased the maximum response to phenylephrine by 60% and resulted in a plasma concentration of 500 ± 180 nmol/L. Amiloride (100µmol/L) caused rightward shifts in concentration-response curves to phenylephrine in vitro. Long-term treatment with hydrochlorothiazide plus amiloride did not significantly increase the maximum response to phenylephrine (+13%). Reverse transcriptase polymerase chain reaction did not detect the NaCl cotransporter in mouse CC. Hydrochlorothiazide did not change Rho-kinase activity, whereas amiloride decreased it in vitro and ex vivo (approximately 18% and 24% respectively). A 40% decrease in Rock1 expression also was observed after long-term treatment with hydrochlorothiazide plus amiloride. CONCLUSION: Hydrochlorothiazide potentiates contraction of smooth muscle from mouse CC. These findings could explain why diuretics such as hydrochlorothiazide are associated with erectile dysfunction.
RESUMO
A rapid, sensitive and specific method for quantifying piracetam in human plasma using Piracetam d-8 as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by one-step precipitation of protein using an acetonitrile (100%). The extracts were analyzed by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS/MS). The method had a chromatographic run time of 3.8 min and a linear calibration curve over the range 0.5-50 µg/mL (r > 0.99). This LC-MS-MS procedure was used to assess the bioavailability of two piracetam formulations: piracetam + l-carnitine (Piracar®; 270/330 mg tablet) and piracetam (Nootropil®; 800 mg tablet) in healthy volunteers of both sexes. The geometric means with corresponding 90% confidence interval (CI) for test/reference percentage ratios were 88.49% (90% CI = 81.19 - 96.46) for peak concentration/dose and 102.55% (90% CI = 100.62 - 104.51) for AUCinf /dose. The limit of quantitation of 0.5 µg/mL is well suited for pharmacokinetic studies in healthy volunteers. It was concluded that piracetam (Piracar®; 270/330 mg tablet) has a bioavailability equivalent to the piracetam (Nootropil®; 800 mg tablet) formulation with regard to both the rate and the extent of absorption.
Assuntos
Carnitina/sangue , Fármacos Neuroprotetores/sangue , Piracetam/sangue , Administração Oral , Adolescente , Adulto , Disponibilidade Biológica , Carnitina/administração & dosagem , Cromatografia Líquida de Alta Pressão/métodos , Feminino , Voluntários Saudáveis , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Fármacos Neuroprotetores/administração & dosagem , Piracetam/administração & dosagem , Espectrometria de Massas por Ionização por Electrospray/métodos , Comprimidos , Espectrometria de Massas em Tandem/métodos , Adulto JovemRESUMO
ABCC1 and ABCG2 are two transporters associated with multi-drug resistance to cancer chemotherapy. Ouabain is a cardiotonic steroid, currently considered as a hormone associated with arterial hypertension. Previous studies have suggested that ouabain can modulate ABCB1 and ABCC1 expression in cancer and renal cell lines. The present study investigated the effects of physiological concentrations of ouabain on the expression and activity of ABCC1 and ABCG2 in two human breast cancer cell lines, MCF7 and MDA-MB-231, the first known to be responsive to estrogens. Cell viability and proliferation assays showed that 1 µM ouabain reduced proliferation of MCF7, but not if MDA-MB-231 cells. On the other hand, 10 nM ouabain increased proliferation of MDA-MB-231, but not of MCF7 cells. Ouabain (10 nM) prevented the cytotoxic effects of doxorubicin in MCF7 cells, but not in MDA-MB-231 cells. Treatment of cells under different ouabain concentrations for 24 h did not cause any significant effects in the expression of ABCG2 or ABCC1 in either cell line. However, the activity of ABCC1 was increased when MCF7 and MDA-MB-231 cells were treated with 10 mM and 1 nM ouabain respectively. These results claim attention to the possibility that breast cancer patients with high levels of endogenous ouabain may have different responses to chemotherapy.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Ouabaína/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Cardiotônicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Imunofluorescência , Humanos , Células Tumorais CultivadasRESUMO
Informed consent is recognized as a primary ethical requirement to conduct research involving humans. In the investigations with the use of human biological material, informed consent (IC) assumes a differentiated condition on account of the many future possibilities. This work presents suitable alternatives for IC regarding the storage and use of human biological material in research, according to new Brazilian regulations. Both norms - Resolution 441/11 of the National Health Council, approved on 12 May 2011, and Ordinance 2.201 (NATIONAL GUIDELINES FOR BIOREPOSITORIES AND BIOBANKS OF HUMAN BIOLOGICAL MATERIAL FOR RESEARCH PURPOSE) of the Brazil Ministry of Health, approved on 14 September 2011 - state that the consent of subjects for the collection, storage and use of samples stored in Biobanks is necessarily established by means of a Free and Informed Consent Form (ICF). In order to obtain individual and formal statements, this form should contain the following two mutually exclusive options: an explanation about the use of the stored material in each research study, and the need for new consent or the waiver thereof when the material is used for a new study. On the other hand, ICF suitable for Biorepositories must be exclusive and related to specific research. Although Brazilian and international regulations identify the main aspects to be included in the IC, efforts are still necessary to improve the consent process, so that the document will become a bond of trust between subject and researcher.
Assuntos
Pesquisa Biomédica/ética , Confidencialidade/ética , Consentimento Livre e Esclarecido/ética , Manejo de Espécimes/ética , Bancos de Tecidos/ética , Brasil , Humanos , Bancos de Tecidos/legislação & jurisprudência , Bancos de Tecidos/tendênciasRESUMO
Angiotensin II (Ang II) stimulates the proximal tubule Na(+)-ATPase through the AT(1) receptor/phosphoinositide phospholipase Cbeta (PI-PLCbeta)/protein kinase C (PKC) pathway. However, this pathway alone does not explain the sustained effect of Ang II on Na(+)-ATPase activity for 30 min. The aim of the present work was to elucidate the molecular mechanisms involved in the sustained effect of Ang II on Na(+)-ATPase activity. Ang II induced fast and correlated activation of Na(+)-ATPase and PKC activities with the maximal effect (115%) observed at 1 min and sustained for 30 min, indicating a pivotal role of PKC in the modulation of Na(+)-ATPase by Ang II. We observed that the sustained activation of PKC by Ang II depended on the sequential activation of phospholipase D and Ca(2+)-insensitive phospholipase A(2), forming phosphatidic acid and lysophosphatidic acid, respectively. The results indicate that PKC could be the final target and an integrator molecule of different signaling pathways triggered by Ang II, which could explain the sustained activation of Na(+)-ATPase by Ang II.
Assuntos
Angiotensina II/farmacologia , Fosfolipase D/metabolismo , Fosfolipases A2/metabolismo , Proteína Quinase C/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Lisofosfolipídeos/metabolismo , Membranas/efeitos dos fármacos , Ácidos Fosfatídicos/farmacologia , Sus scrofa , Fatores de TempoRESUMO
BACKGROUND: Propofol (2,6-diisopropylphenol) has been shown to protect several organs, including the kidneys, from ischemia-reperfusion (I-R)-induced injury. Although propofol affects adenosine triphosphate-sensitive potassium (K(ATP)) channels in nonrenal tissues, it is still not clear by which mechanisms propofol protects renal cells from such damage. In this study, we investigated whether propofol induces renal preconditioning through renal K(ATP) channels. METHODS: A reversible ATP depletion (antimycin A) followed by restoration of substrate supply in LLC-PK1 cells was used as an in vitro model of renal I-R. Cell viability was assessed by dimethylthiazol-diphenyltetrazol bromide and trypan blue dye exclusion test assays. Apoptosis was evaluated by annexin V-fluorescein isothiocyanate staining by flow cytometry and immunofluorescence. Propofol treatments were initiated at various time intervals: 1 or 24 h before ischemia, only during ischemia, or only during reperfusion. To evaluate the mechanisms of propofol protection, specific K(ATP) channel inhibitors or activators were used in some experiments during propofol pretreatment. RESULTS: Propofol attenuated I-R injury on LLC-PK1 cells when present either 1 or 24 h before initiated I-R, and also during the recovery period, but not when added only during ischemia. Propofol pretreatment significantly protected LLC-PK1 from I-R-induced apoptosis. The protective effect of propofol was prevented by glibenclamide (a sarcolemmal ATP-dependent K(+) channel blocker) and decreased by 5-hydroxidecanoic acid (a mitochondrial ATP-dependent K(+) channel blocker), but it was not modified by diazoxide (a selective opener of ATP-sensitive K(+) channel). CONCLUSION: Propofol protected cells against apoptosis induced by I-R. This protection was probably due to a preconditioning effect of propofol and was, at least in part, mediated by K(ATP) channels.
Assuntos
Canais KATP/agonistas , Nefropatias/prevenção & controle , Rim/efeitos dos fármacos , Propofol/farmacologia , Substâncias Protetoras/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Trifosfato de Adenosina/deficiência , Animais , Antimicina A/farmacologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoproteção , Ácidos Decanoicos/farmacologia , Diazóxido/farmacologia , Glibureto/farmacologia , Hidroxiácidos/farmacologia , Canais KATP/metabolismo , Rim/metabolismo , Rim/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Células LLC-PK1 , Necrose , Bloqueadores dos Canais de Potássio/farmacologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Suínos , Fatores de TempoRESUMO
The molecular mechanisms involved in the Ang-(1-7) [angiotensin-(1-7)] effect on sodium renal excretion remain to be determined. In a previous study, we showed that Ang-(1-7) has a biphasic effect on the proximal tubule Na+-ATPase activity, with the stimulatory effect mediated by the AT1 receptor. In the present study, we investigated the molecular mechanisms involved in the inhibition of the Na+-ATPase by Ang-(1-7). All experiments were carried out in the presence of 0.1 nM losartan to block the AT1 receptor-mediated stimulation. In this condition, Ang-(1-7) at 0.1 nM inhibited the Na+-ATPase activity of the proximal tubule by 54%. This effect was reversed by 10 nM PD123319, a specific antagonist of the AT2 receptor, and by 1 muM GDP[beta-S] (guanosine 5'-[beta-thio]diphosphate), an inhibitor of G protein. Ang-(1-7) at 0.1 M induced [35S]GTP[S] (guanosine 5'-[gamma-[35S]thio]triphosphate) binding and 1 mug/ml pertussis toxin, an inhibitor of G(i/o) protein, reversed the Ang-(1-7) effect. Furthermore, it was observed that the inhibitory effect of Ang-(1-7) on the Na+-ATPase activity was completely reversed by 0.1 microM LY83583, an inhibitor of guanylate cyclase, and by 2 muM KT5823, a PKG (protein kinase G) inhibitor, and was mimicked by 10 nM d-cGMP (dibutyryl cGMP). Ang-(1-7) increased the PKG activity by 152% and this effect was abolished by 10 nM PD123319 and 0.1 microM LY83583. Taken together, these data indicate that Ang-(1-7) inhibits the proximal tubule Na+-ATPase by interaction with the AT2 receptor that subsequently activates the G(i/o) protein/cGMP/PKG pathway.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Angiotensina I/farmacologia , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Receptor Tipo 2 de Angiotensina/metabolismo , Adenosina Trifosfatases/metabolismo , Bloqueadores do Receptor Tipo 2 de Angiotensina II , Animais , Proteínas de Transporte de Cátions/metabolismo , Córtex Renal/efeitos dos fármacos , Córtex Renal/enzimologia , Córtex Renal/metabolismo , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/metabolismo , Transdução de Sinais/efeitos dos fármacos , SuínosRESUMO
Costus spicatus, used in Brazilian traditional medicine to expel kidney stones, contains steroidal saponins with different chemical characteristics. In spite of its popular utilization as potent diuretic, no scientific reports correlate this activity with the chemical constituents of the extract. Therefore, two steroidal saponins (3 beta,22 alpha,25R)-26-(beta-D-glucopyranosyloxy)-2-methoxyfurost-5-en-3-yl O-D-apio-beta-D-furanosyl-(1-->2)-O-[6-deoxy-alpha-L-mannopyranosyl-(1-->4)]-beta-D-glucopyranoside (1) and (3 beta,22 alpha,25R)-spirostan-3-yl O-D-apio-beta-D-furanosyl-(1-->2)-O-[6-deoxy-alpha-L-mannopyranosyl-(1-->4)]-beta-D-glucopyranoside (1a), were isolated from the rhizomes of this plant and their effects on the Na+-ATPase and (Na+ + K+)-ATPase activities of the proximal tubule from pig kidney were evaluated. It was observed that 1 and 1a inhibit specifically the Na+-ATPase activity.
Assuntos
Saponinas/farmacologia , ATPase Trocadora de Sódio-Potássio/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Esteroides/farmacologia , Animais , Terapias Complementares , Costus , Cães , Rim/efeitos dos fármacos , Rim/enzimologia , Rim/fisiologia , Cinética , Fitoterapia , SuínosRESUMO
Trypanosoma cruzi, the protozoan responsible for Chagas disease, employs distinct strategies to invade mammalian host cells. In the present work we investigated the participation of calcium ions on the invasion process using primary cultures of embryonic mice cardiomyocytes which exhibit spontaneous contraction in vitro. Using Fura 2-AM we found that T. cruzi was able to induce a sustained increase in basal intracellular Ca2+ level in heart muscle cells (HMC), the response being associated or not with Ca2+ transient peaks. Assays performed with both Y and CL strains indicated that the changes in intracellular Ca2+ started after parasites contacted with the cardiomyocytes and the evoked response was higher than the Ca2+ signal associated to the spontaneous contractions. The possible role of the extracellular and intracellular Ca2+ levels on T. cruzi invasion process was evaluated using the extracellular Ca2+ chelator EGTA alone or in association with the calcium ionophore A23187. Significant dose dependent inhibition of the invasion levels were found when intracellular calcium release was prevented by the association of EGTA +A23187 in calcium free medium. Dose response experiments indicated that EGTA 2.5 mM to 5 mM decreased the invasion level by 15.2 to 35.1% while A23187 (0.5 M) alone did not induce significant effects (17%); treatment of the cultures with the protease inhibitor leupeptin did not affect the endocytic index, thus arguing against the involvement of leupeptin sensitive proteases in the invasion of HMC.
Assuntos
Cálcio/metabolismo , Miócitos Cardíacos/parasitologia , Trypanosoma cruzi/fisiologia , Animais , Células Cultivadas , Quelantes/farmacologia , Citosol/parasitologia , Ácido Egtázico/farmacologia , Camundongos , Microscopia de Fluorescência , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Sarcolema/parasitologia , Fatores de Tempo , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Trypanosoma cruzi, the protozoan responsible for Chagas disease, employs distinct strategies to invade mammalian host cells. In the present work we investigated the participation of calcium ions on the invasion process using primary cultures of embryonic mice cardiomyocytes which exhibit spontaneous contraction in vitro. Using Fura 2-AM we found that T. cruzi was able to induce a sustained increase in basal intracellular Ca2+ level in heart muscle cells (HMC), the response being associated or not with Ca2+ transient peaks. Assays performed with both Y and CL strains indicated that the changes in intracellular Ca2+ started after parasites contacted with the cardiomyocytes and the evoked response was higher than the Ca2+ signal associated to the spontaneous contractions. The possible role of the extracellular and intracellular Ca2+ levels on T. cruzi invasion process was evaluated using the extracellular Ca2+ chelator EGTA alone or in association with the calcium ionophore A23187. Significant dose dependent inhibition of the invasion levels were found when intracellular calcium release was prevented by the association of EGTA +A23187 in calcium free medium. Dose response experiments indicated that EGTA 2.5 mM to 5 mM decreased the invasion level by 15.2 to 35.1 percent while A23187 (0.5 æM) alone did not induce significant effects (17 percent); treatment of the cultures with the protease inhibitor leupeptin did not affect the endocytic index, thus arguing against the involvement of leupeptin sensitive proteases in the invasion of HMC
Assuntos
Animais , Camundongos , Cálcio , Trypanosoma cruzi , Células Cultivadas , Quelantes , Citosol , Ácido Egtázico , Microscopia de Fluorescência , Sarcolema , Fatores de Tempo , Trypanosoma cruziRESUMO
The cystic fibrosis transmembrane regulator (CFTR) is a Cl- channel. Mutations of this transporter lead to a defect of chloride secretion by epithelial cells causing the Cystic Fibrosis disease (CF). In spite of the high expression of CFTR in the kidney, patients with CF do not show major renal dysfunction, but it is known that both the urinary excretion of drugs and the renal capacity to concentrate and dilute urine is deficient. CFTR mRNA is expressed in all nephron segments and its protein is involved with chloride secretion in the distal tubule, and the principal cells of the cortical (CCD) and medullary (IMCD) collecting ducts. Several studies have demonstrated that CFTR does not only transport Cl- but also secretes ATP and, thus, controls other conductances such as Na+ (ENaC) and K+ (ROMK2) channels, especially in CCD. In the polycystic kidney the secretion of chloride through CFTR contributes to the cyst enlargement. This review is focused on the role of CFTR in the kidney and the implications of extracellular volume regulators, such as hormones, on its function and expression.
Assuntos
Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Rim/metabolismo , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/isolamento & purificação , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Fibrose Cística/fisiopatologiaRESUMO
Malpighian tubule of Rhodnius sp. express two sodium pumps: the classical ouabain-sensitive (Na+ + K+)ATPase and an ouabain-insensitive, furosemide-sensitive Na+-ATPase. In insects, 5-hydroxitryptamine is a diuretic hormone released during meals. It inhibits the (Na+ + K+)ATPase and Na+ -ATPase activities indicating that these enzymes are involved in fluid secretion. Furthermore, in Rhodnius neglectus, proximal cells of Malpighian tubule exposed to hyperosmotic medium, regulate their volume through a mechanism called regulatory volume increase. This regulatory response involves inhibition of the (Na+ + K+)ATPase activity that could lead to accumulation of active osmotic solute inside the cell, influx of water and return to the normal cell volume. Adenosine, a compound produced in stress conditions, also inhibits the (Na+ + K+)ATPase activity. Taken together these data indicate that (Na+ + K+)ATPase is a target of the regulatory mechanisms of water and ions transport responsible for homeostasis in Rhodnius sp.
Assuntos
Animais , Insetos Vetores/metabolismo , Túbulos de Malpighi/enzimologia , Rhodnius/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/fisiologiaRESUMO
The present paper summarizes new approaches regarding the progress done to the understanding of the interaction of Trypanosoma cruzi-cardiomyocytes. Mannose receptors localized at the surface of heart muscle cell are involved in binding and uptake of the parasite. One of the most striking events in the parasite-heart muscle cells interaction is the disruption of the actin cytoskeleton. We have investigated the regulation of the actin mRNA during the cytopathology induced in myocardial cells by the parasite. T. cruzi invasion increases calcium resting levels in cardiomyocytes. We have previously shown that Ca2+ ATPase of the sarcoplasmic reticulum (SERCA) is involved in the invasion of T. cruzi in cardiomyocytes. Treating the cells with thapsigargin, a drug that binds to all SERCA ATPases and causes depletion of intracellular calcium stores, we found a 75 per cent inhibition in the T. cruzi-cardiomyocytes invasion.