Effects of renal denervation on endogenous ouabain in spontaneously hypertensive rats

Purpose: To investigate the role of renal denervation (RDN) on endogenous ouabain (EO) secretion in spontaneously hypertensive rats (SHR). Methods: Sixteen 12-week-old male SHR were randomly separated into the renal denervation group (RDNX group) and sham operation group (sham group), and eight age-matched Wistar Kyoto rats (WKY) were served as control group. EO concentrations, the Na+- K+-ATPaseactivity, and the expression of Na+-K+-ATPase were assessed. Results: EO levels in serum, kidneys and hypothalamus of sham group were higher than in RDNX group (p < 0.05). Renal Na+-K+-ATPase activity subjected to denervation surgery showed significantly reduction when compared with the sham groups (p < 0.05). A positive correlation existed between norepinephrine (NE) content and Na+-K+-ATPase activity in the kidney (r2 = 0.579). Renal Na+-K+-ATPase α1 subunit mRNA expression was down-regulated in the RDNX group compared with the sham group (P < 0.05), while renal Na+-K+-ATPase α1 subunit mRNA expression was no statistical significance between the groups (P = 0.63). Immunohistochemical analysis showed that there were significant differences in the renal expression of Na+-K+-ATPasebetween the three groups (P < 0.05). Conclusions: These experiments demonstrate that RDN exerted an anti-hypertensive effect with reduction of EO levels and Na+-K+-ATPase activity and Na+-K+-ATPase α1 subunit expression of kidney in SHR.

In vitro and in silico evaluation of the schistosomicidal activity of eugenol derivatives using biochemical, molecular, and morphological tools

Background Eugenol shows both antibacterial and antiparasitic activities, suggesting that it might be evaluated as an option for the treatment of praziquantel-resistant schistosome. Methods The in vitro activities of three eugenol derivatives (FB1, FB4 and FB9) on adult worms from Schistosoma mansoni were examined by fluorescence and scanning electron microscopy to analyze effects on the excretory system and integument damage, respectively. Biochemical tests with verapamil (a calcium channel antagonist) and ouabain (a Na+/K+-ATPase pump inhibitor) were used to characterize eugenol derivative interactions with calcium channels and the Na+/K+-ATPase, while in silico analysis identified potential Na+/K+-ATPase binding sites. Results The compounds showed effective doses (ED50) of 0.324 mM (FB1), 0.167 mM (FB4), and 0.340 mM (FB9). In addition, FB4 (0.322 mM), which showed the lowest ED50, ED90 and ED100 (p < 0.05), caused the most damage to the excretory system and integument, according to both fluorescence and scanning electron microscopy analysis. The death of adult worms was delayed by ouabain treatment plus FB1 (192 versus 72 hours) and FB9 (192 versus 168 hours), but the response to FB4 was the same in the presence or absence of ouabain. Besides, no changes were noted when all of the eugenol derivatives were combined with verapamil. Moreover, FB1 and FB9 inhibited Na+/K+-ATPase activity according to in silico analysis but FB4 did not show a time-dependent relationship and may act on targets other than the parasite Na+/K+-ATPase. Conclusion Eugenol derivatives, mainly FB4 when compared to FB1 and FB9, seem to act more effectively on the integument of adult S. mansoni worms.(AU)

Inhibition of Na+, K+ -ATPase with ouabain is detrimental to equine blastocysts

Although equine blastocysts ≤ 300 µm in diameter can be successfully vitrified, larger equine blastocysts are not good candidates for cryopreservation. As Na+, K+-ATPase is involved in maintaining blastocyst expansion, perhaps inhibition of this enzyme would be a viable method of reducing blastocyst diameter prior to cryopreservation. Objectives were to evaluate effects of ouabain-induced inhibition of Na+, K+-ATPase in equine blastocysts. Sixteen mares were ultrasonographically monitored, given deslorelin acetate to induce ovulation, and inseminated. Embryos (D7 and D9) were harvested and Na+, K+-ATPase inhibited for 1 or 6 h by exposure to 10-6 M ouabain, either natural ouabain or conjugated to fluorescein (OuabainFL), during incubation at 37° C. Evaluations included morphometric characteristics (bright field microscopy) and viability (Hoescht 33342 + propidium iodide). Blastocysts incubated for 6 h in Holding medium + ouabain (n=3) had, on average, a 45.7% reduction in diameter, with adverse morphologic features and no re-expansion after subsequent incubation in Holding medium for 12 h. In subsequent studies, even a 1-h exposure to Ouabain or OuabainFL, caused similar reductions, namely 38.7 ± 6.7% (n=5) and 33.6 ± 3.3% (n=7) for D7 and D9 blastocysts, respectively. Ouabain binding was confirmed after OuabainFL exposition and all embryos (n=12) lost viability. We concluded that Na+, K+-ATPase inhibition with ouabain caused death of equine blastocysts and therefore was not a viable method of reducing blastocyst size prior to cryopreservation.

Inhibition of Na+, K+ -ATPase with ouabain is detrimental to equine blastocysts

Although equine blastocysts ≤ 300 µm in diameter can be successfully vitrified, larger equine blastocysts are not good candidates for cryopreservation. As Na+, K+-ATPase is involved in maintaining blastocyst expansion, perhaps inhibition of this enzyme would be a viable method of reducing blastocyst diameter prior to cryopreservation. Objectives were to evaluate effects of ouabain-induced inhibition of Na+, K+-ATPase in equine blastocysts. Sixteen mares were ultrasonographically monitored, given deslorelin acetate to induce ovulation, and inseminated. Embryos (D7 and D9) were harvested and Na+, K+-ATPase inhibited for 1 or 6 h by exposure to 10-6 M ouabain, either natural ouabain or conjugated to fluorescein (OuabainFL), during incubation at 37° C. Evaluations included morphometric characteristics (bright field microscopy) and viability (Hoescht 33342 + propidium iodide). Blastocysts incubated for 6 h in Holding medium + ouabain (n=3) had, on average, a 45.7% reduction in diameter, with adverse morphologic features and no re-expansion after subsequent incubation in Holding medium for 12 h. In subsequent studies, even a 1-h exposure to Ouabain or OuabainFL, caused similar reductions, namely 38.7 ± 6.7% (n=5) and 33.6 ± 3.3% (n=7) for D7 and D9 blastocysts, respectively. Ouabain binding was confirmed after OuabainFL exposition and all embryos (n=12) lost viability. We concluded that Na+, K+-ATPase inhibition with ouabain caused death of equine blastocysts and therefore was not a viable method of reducing blastocyst size prior to cryopreservation.(AU)

Na/K-ATPase and Regulation of Sperm Function

A standard bull breeding soundness evaluation (BBSE) identifies bulls with semen that is grossly abnormal. Nonetheless, semen samples classified as satisfactory based on these traditional approaches differ in fertility; perhaps there are submicroscopic differences in sperm characteristics affecting fertility. Therefore, a better understanding of molecular regulation of sperm function could promote development of novel, evidence-based approaches to predict male fertility. Recently the α4 isoform of Na/K-ATPase (ATP1A4) has received considerable attention, due to its testisspecific expression in post-meiotic germ cells and mature sperm, in addition to its regulation of sperm motility and capacitation. Using fresh bull sperm, we determined that ATP1A4 resided in specialized microdomains (raft and non-raft) of the sperm plasma membrane and activated specific signaling (caveolin-1, EGFR, Src, ERK1/2) molecules during sperm capacitation. Furthermore, ATP1A4 was the predominant isoform responsible for total Na/K-ATPase activity in capacitated sperm. Despite the widely accepted dogma of transcriptional/translational quiescence, bovine sperm translated ATP1A4 mRNA on mitochondrial or mitochondrial-type ribosomes, increasing their content and activity during capacitation. Proteomic analysis of raft and non-raft fractions revealed a significant interaction between ATP1A4 and plakoglobin, a member of the β-catenin family of proteins involved in cell adhesion, in the equatorial segment of capacitated sperm, suggesting a potential role in sperm-oolemma fusion. In frozen-thawed sperm, ATP1A4 content and activity was greater in high- versus low-fertility bulls. Additionally, ATP1A4-induced increases in ROS, calcium, actin polymerization and tyrosine phosphorylation were also involved in regulating post-thaw sperm function in these bulls. Overall, results demonstrated that ATP1A4 had unique roles in controlling several aspects of sperm physiology, acting through well-established enzyme activity and signaling functions. Consequently, isoforms of Na/K-ATPase are potential biomarkers for male fertility.

Na/K-ATPase and Regulation of Sperm Function

A standard bull breeding soundness evaluation (BBSE) identifies bulls with semen that is grossly abnormal. Nonetheless, semen samples classified as satisfactory based on these traditional approaches differ in fertility; perhaps there are submicroscopic differences in sperm characteristics affecting fertility. Therefore, a better understanding of molecular regulation of sperm function could promote development of novel, evidence-based approaches to predict male fertility. Recently the α4 isoform of Na/K-ATPase (ATP1A4) has received considerable attention, due to its testisspecific expression in post-meiotic germ cells and mature sperm, in addition to its regulation of sperm motility and capacitation. Using fresh bull sperm, we determined that ATP1A4 resided in specialized microdomains (raft and non-raft) of the sperm plasma membrane and activated specific signaling (caveolin-1, EGFR, Src, ERK1/2) molecules during sperm capacitation. Furthermore, ATP1A4 was the predominant isoform responsible for total Na/K-ATPase activity in capacitated sperm. Despite the widely accepted dogma of transcriptional/translational quiescence, bovine sperm translated ATP1A4 mRNA on mitochondrial or mitochondrial-type ribosomes, increasing their content and activity during capacitation. Proteomic analysis of raft and non-raft fractions revealed a significant interaction between ATP1A4 and plakoglobin, a member of the β-catenin family of proteins involved in cell adhesion, in the equatorial segment of capacitated sperm, suggesting a potential role in sperm-oolemma fusion. In frozen-thawed sperm, ATP1A4 content and activity was greater in high- versus low-fertility bulls. Additionally, ATP1A4-induced increases in ROS, calcium, actin polymerization and tyrosine phosphorylation were also involved in regulating post-thaw sperm function in these bulls. Overall, results demonstrated that ATP1A4 had unique roles in controlling several aspects of sperm physiology, acting through well-established enzyme activity and signaling functions. Consequently, isoforms of Na/K-ATPase are potential biomarkers for male fertility.(AU)

EFEITOS DA OLEANDRINA, OUABAÍNA e DIGOXINA NO TRATAMENTO DA INSUFICIÊNCIA CARDÍACA INDUZIDA POR DOXORRUBICINA

A insuficiência cardíaca congestiva atinge milhões de pessoas em todo o mundo e também é uma realidade na medicina veterinária de pequenos animais. Novas modalidades terapêuticas são importantes para melhorar a qualidade de vida dos portadores dessa síndrome. Para avaliar o potencial terapêutico dos glicosídeos cardioativos oleandrina e ouabaína e compará-los à digoxina, glicosídeo cardioativo com uso bem estabelecido, foram usados 20 ratos Wistar, distribuídos em quatro grupos, com cinco animais em cada grupo. Os animais foram desafiados com uma única aplicação intraperitoneal de doxorrubicina, na dose de 12mg/kg, e tratados com solução salina e com os glicosídeos oleandrina, ouabaína e digoxina, na dose de 50g/kg, durante sete dias. Foram submetidos a exames de ecocardiografia, eletrocardiografia, hematológicos e bioquímicos séricos. Todos os animais apresentaram insuficiência cardíaca congestiva, constatada por redução da fração de ejeção. Oleandrina e digoxina foram capazes de reduzir significativamente (p<0,05) o remodelamento excêntrico causado pela doxorrubicina, porém foram significativamente inferiores (p<0,05) ao grupo controle na manutenção do volume sistólico e do volume do ventrículo esquerdo na diástole. Demais parâmetros cardíacos avaliados não apresentaram diferenças significativas. Todos os animais apresentaram aumento do número de eritrócitos, aumento da duração do complexo QRS no ECG e, necrose miocárdica na análise histopatológica. Conclui-se que os glicosídeos oleandrina, ouabaína e digoxina, na posologia empregada, não apresentaram potencial terapêutico para tratamento de insuficiência cardíaca congestiva causada pela doxorrubicina

Congestive heart failure affects millions of people worldwide and is also a reality in small animal veterinary medicine. New therapeutic modalities are important to improve the quality of life of patients with this syndrome. In order to evaluate the therapeutic potential of cardioactive glycosides oleandrin and ouabain, and compare them to digoxin, glycoside with well-established use, 20 Wistar rats were used in the study, distributed into four groups with five animals in each group. The animals were challenged with a single intraperitoneal application of doxorubicin, at a dose of 12mg / kg, and treated with saline solution and the glycosides oleandrin, ouabain and digoxin, at a dose of 50g/kg for seven days. They underwent echocardiography, electrocardiography, hematological and biochemical tests. All animals presented congestive heart failure, which was verified by a reduction in the ejection fraction. Oleandrin and digoxin were able to significantly reduce (p <0.05) the eccentric remodeling caused by doxorubicin. Oleandrin and digoxin were significantly lower (p <0.05) than control group in maintaining systolic volume and left ventricular volume in diastole. Other cardiac parameters evaluated did not show significant statistical differences. All animals showed an increase in erythrocyte count, an increase in the duration of the QRS complex on the ECG and myocardial necrosis in the histopathological analysis. It is concluded that the glycosides oleandrin, ouabain and digoxin in the used dosage, do not present therapeutic potential for the treatment of congestive heart failure caused by doxorubicin

Presynaptic neuromuscular action of a methanolic extract from the venom of Rhinella schneideri toad

Rhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity. The neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording in response to indirect stimulation and for electrophysiological measurements.(AU)

Associação entre a Na+, K+-ATPase e a fertilidade de amostras seminais descongeladas de touros Angus

Na atualidade, um dos desafios da reprodução bovina é identificar de maneira eficiente touros que apresentam elevada fertilidade, uma vez que a avaliação andrológica tradicional pode não diferenciar de forma precisa o potencial fertilizante de um reprodutor, levando a redução nas taxas de prenhez e prejuízos financeiros. Desta maneira, a Na+, K+-ATPase tem sido um biomarcador potencial da fertilidade, devido a suas atribuições específicas na célula espermática e relatos de correlação com a fertilidade de touros Holandeses. No entanto devido a peculiaridades de manejo reprodutivo da pecuária brasileira de corte e pela ausência de pesquisas envolvendo a Na+, K+-ATPase em bovinos com esta aptidão, o presente estudo foi desenvolvido a fim de determinar a associação entre a atividade da Na+, K+-ATPase em espermatozoides descongelados de touros Angus classificados como de fertilidade superior (FS) e normal (FN) após inseminação artificial em tempo fixo (IATF). Foram utilizadas três diferentes partidas de amostras comerciais de touros de FS (n = 4) e FN (n = 4). Realizou-se a avaliação da cinemática através do sistema CASA, da viabilidade espermática através da microscopia de fluorescência e da expressão de Na+, K+-ATPase através da citometria de fluxo, imediatamente após a descongelação (0h) e após 2h de incubação (37 °C), enquanto a atividade da Na+, K+-ATPase foi determinada mensurando-se a liberação de fosfato inorgânico (iP) nas amostras tratadas ou não com inibidor específico (ouabaína, 4×10-4 M), sendo normalizada em função da proteína total de cada amostra. Dentro do mesmo tempo de incubação não houve diferença (P > 0,05) na viabilidade, cinemática e na expressão da Na+, K+-ATPase entre touros FS e FN. Os parâmetros cinemáticos de LIN e VCL não foram influenciados pela incubação nas amostras de FS e FN, respectivamente. Houve uma tendência (P = 0,06) de maior atividade enzimática da Na+, K+-ATPase em amostras de touros de FS. Em conclusão, diferenças na cinemática e viabilidade espermática não são detectadas entre touros Angus de FS e FN imediatamente após o descongelamento. Diferentes respostas nos parâmetros de velocidade pós-incubação são detectadas em touros de FS e FN que podem estar relacionadas a atividade da Na+, K+-ATPase, sem influência na sua expressão.

Currently, one of the challenges on bovine reproduction is to efficiently identify high fertility bulls, since traditional andrological evaluation may not precisely differ the fertilizing potential of a sire, leading to reduced pregnancy rates and economic losses. Therefore, the Na+, K+-ATPase has been considered a potential fertility marker, due to its sperm specific properties and reports o correlation with Holstein bull fertility. However, specificities on reproductive management of Brazilian beef herds associated to the lack of information regarding Na+, K+-ATPase in beef bulls, the present study was performed aiming to determine association between Na+, K+-ATPase activity in thawed Angus bull sperm scored as being a high fertility (HF) or normal fertility (NF) after fixed time artificial insemination (FTAI). Samples from three different commercial batches of bulls of HF (n=4) or NF (n=4) were used. Sperm kinematics was evaluated with CASA system, sperm viability was assessed by fluorescent microscopy, and expression of Na+, K+-ATPase on the sperm surface by flow citometry, immediately post-thaw (0 h) and after 2 h of incubation (37 °C). Na+, K+-ATPase activity was determined measuring the releasing of inorganic phosphate (iP) in the samples treated or not with a specific inhibitor (ouabain, 4×10-4 M), normalized by total protein of each sample. Within the same incubation time, there was no difference (P > 0.05) on sperm viability, kinematic parameters, and the expression of Na+, K+-ATPase between HF and NF bulls. Kinematic parameters of LIN and VCL were not influenced (P > 0.05) by incubation time in samples from HF and NF, respectively. There was a tendency (P = 0.06) of higher Na+, K+-ATPase enzymatic activity in HF bull samples. In conclusion, differences on kinematic and sperm viability are not detected between HF and NF Angus bulls immediately post-thaw. Different responses on velocity parameters post-incubation were detected in HF and NF bulls that might be related to Na+, K+-ATPase activity, without influence of Na+, K+-ATPase expression.

Presynaptic neuromuscular action of a methanolic extract from the venom of Rhinella schneideri toad

Rhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity. The neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording – in response to indirect stimulation – and for electrophysiological measurements.

Influência do bloqueio da Na+, K+-ATPase em blastocistos equinos

Atualmente, um dos desafios na reprodução equina é a baixa taxa de sobrevivência de embriões criopreservados com diâmetro > 300 m, usando métodos de vitrificação ou clássicos. Pesquisas sobre a influência da bomba de sódio e potássio (Na+, K+-ATPase) no desenvolvimento embrionário de equinos têm mais de 30 anos e hoje se sabe que participa da cavitação embrionária, modifica o gradiente iônico das células trofoblásticas, além de influenciar a formação, distribuição e permeabilidade das tight junctions (TJ) entre células trofoblásticas. Devido aos diversos tipos de isoformas da Na +, K+-ATPase, a depender das subunidades que a compõe, e a peculiaridades do desenvolvimento embrionário nas diferentes espécies, esse trabalho é caracterizado por uma revisão de literatura, para demonstrar os resultados dos principais estudos na área. Apresenta os resultados de um estudo que foi desenvolvido com o objetivo de buscar um meio capaz de reduzir o diâmetro do embrião equino a campo, para apoiar a vitrificação do blastocisto. Embriões equinos foram recuperados e submetidos a inibição da Na+, K+-ATPase por 1 ou 6 h, utilizando ouabaína isolada ou conjugada à sonda fluorescente (OuabaínaFL), durante incubação a 37° C. Os embriões foram avaliados quanto a características morfométricas (microscopia em campo claro) e de viabilidade (Hoechst 33342 + iodeto de propídeo). Acredita-se que este tenha sido o primeiro estudo que objetivou avaliar primariamente os efeitos da inibição da Na+, K+-ATPase com ouabaína, um inibidor específico, sobre embriões equinos. Observou-se que a inibição da Na+, K+-ATPase com ouabaína é capaz de reduzir o diâmetro dos blastocistos equinos, independetemente do dia de desenvolvimento (D7 ou D9), tendo sua confirmada a capacidade de ligação aos embriões a partir de estudos com ouabaína conjugada a sonda fluorescente. Todavia, a inibição da Na+, K+-ATPase compromente a viabilidade do embrião, não sendo recomendada a sua adoção com estratégia para redução do diâmetro de blastocistos equinos.

Currently, one of the challenges on equine reproduction is the low survival rate of horse embryos cryopreserved with diameter > 300 m, using either vitrification or classic methods. Researches about the influence of sodium pump and potassium (Na+, K+-ATPase) on horse embryo development have more than 30 years and nowadays it is known that participates on embryo cavitation, changes ionic gradient of trophoblastic cells, as well as influences formation, distribution and permeability of tight junctions (TJ) between trophoblastic cells. Due to many Na+, K+-ATPase isoforms, depending on comprising subunits, as well as peculiarities on embryo development depends on species, this work is characterizied by a literature review to demonstrate the main results in the field of research. It is also presented the results of a study that aimed to develop a medium able to reduce horse embryo diameter in the field, to support horse blastocyst vitrification. Horse embryos were hasvested and subjected to Na+, K+-ATPase inhibition for 1 or 6 h, using ouabain isolated or conjugates to fluorescent probe (OuabaínaFL), during incubation at 37 ° C. Embryos were assessed for morphometric characteristics (bright field microscopy) and viability (Hoechst 33342 + propridium iodide). To our knowledge, this is the first study that primarly aimed to evaluate the influence of Na+, K+-ATPase inhibition with ouabain, a specific inhibitor, on the horse embryos. It was observed that Na+, K+-ATPase inhibition with ouabain is able to reduce horse blastocyst diameter, in spite of embryo development (D7 ou D9), and ouabain binding to blastocysts were confirmed after embryo exposition to fluorescent conjugated ouabain. However, Na+, K+-ATPase inhibiton compromised embryo viability and it is not recommended as a strategy to reduce horse blastocyst diameter.

Efeitos dos Glicosídeos Cardioativos ( digoxina ouabaína e oleandrina ) na Fisiologia Cardiorrenal em Ratos Wistar Hígidos

Os glicosídeos cardioativos (GCs) são substâncias que atuam na bomba de sódio e potássio (NKA) do coração exercendo atividade inotrópica. O GC mais conhecido é a digoxina, empregado até hoje no tratamento da insuficiência cardíaca (IC). No entanto, sua restrita margem de segurança impulsiona a descoberta de novas substâncias cardioativas. Nesse âmbito, pouco se sabe sobre a atuação cardiovascular da oleandrina e ouabaína. O objetivo deste trabalho foi avaliar os efeitos comparativos dos GCs na fisiologia cardiorrenal. Inicialmente, foi realizado estudo da posologia da digoxina determinando-se que a dose efetiva capaz de alterar a contratilidade dos cardiomiócitos sem causar arritmias graves é de 50ug/kg via SC por 21 dias. Determinaram-se os valores de referência para os parâmetros eletrocardiográficos (ECG) e a melhor fórmula de correção do intervalo QT para ratos Wistar. Estabeleceu-se um protocolo anestésico para realização de ECG com isoflurano e determinação dos valores médios, desviopadrão, mínimo e máximo dos principais parâmetros: frequência cardíaca (FC), duração e amplitude da onda P, duração dos intervalos PR e QT, duração do complexo QRS, amplitude das ondas R e T. Também foi feita a avaliação comparativa das fórmulas de QT, onde a fórmula de Hodges apresentou melhor correlação com a FC. E, finalmente, avaliaram-se os efeitos comparativos da digoxina, ouabaína e oleandrina na fisiologia cardiorrenal. A análise comparativa entre os GCs revelou importantes diferenças quanto à sua ação no sistema cardiovascular. Os GCs alteraram a série vermelha do hemograma, o que foi comprovado pela presença de equinócitos observados pela microscopia de varredura. Os animais que receberam oleandrina apresentaram lesões cardíacas mais graves observadas pela maior atividade sérica de LDH, aumento relativo da CK-MB necrose focal moderada de cardiomiócitos visto na microscopia óptica; ruptura, encurtamento e desorganização das fibras cardíacas como observado na microscopia eletrônica de varredura. Os três tratamentos provocaram aumento relativo da creatinina e discretas lesões observadas na microscopia óptica renal. A expressão da troponina I e isoforma alfa-1 não foram alteradas, no entanto os níveis proteicos de BNP e alfa- 2 foram superiores nos grupos que receberam oleandrina e ouabaína em relação ao grupo digoxina. Todos os GCs afetaram a produção de ROS, sem no entanto causar peroxidação lipídica, por meio da ativação de diferentes vias antioxidantes. Conclui-se que os efeitos dos GCs são variáveis, possivelmente pela diferença entre a afinidade e inibição da bomba NKA.

Cardioactive glycosides (GCs) are substances that act on the sodium and potassium pump of the heart (NKA) exerting inotropic activity. The best known GC is digoxin, used to date in the treatment of heart failure (HF). However, its narrow range of safety drives the discovery of new cardioactive substances. In this context, little is known about cardiovascular performance of oleandrin and ouabain. The aim of this study was to evaluate the comparative effects of GCs on cardiorenal physiology. Initially, a digoxin dosage study was performed, determining that the effective dose capable of altering the contractility of cardiomyocytes without causing serious arrhythmias is 50 ug/kg via SC for 21 days. The reference values for the electrocardiographic parameters (ECG) and the best QT interval correction formula were determined for especially Wistar rats. An anesthetic protocol was established for ECG with isoflurane and determination of mean values, standard deviation, minimum and maximum of the main parameters: heart rate (HR), duration and amplitude of P wave, duration of PR and QT intervals, duration of the QRS complex, amplitude of the R and T waves. The comparative evaluation of the QT formulas was also performed, where the Hodges formula presented a better correlation with the HR. Finally, the comparative effects of digoxin, ouabain, and oleandrin on the cardiorenal system were evaluated. The comparative analysis between GCs revealed important differences in their action on the cardiovascular system. GCs altered the red blood cell series, which was confirmed by the presence of echinocytes observed by scanning microscopy. Animals receiving oleandrin had more severe cardiac lesions observed by increased serum LDH activity, relative increase of CKMB, and moderate focal necrosis of cardiomyocytes seen in light microscopy; rupture, shortening and disorganization of cardiac fibers as observed in scanning electron microscopy. The three treatments caused a relative increase of the creatinine and discrete lesions observed in renal optic microscopy. The expression of troponin I and alpha-1 isoform were not altered, however, the protein levels of BNP and alpha-2 were higher in the groups that received oleandrin and ouabain in relation to digoxin group. All GCs affected the production of ROS, without causing lipid peroxidation, through the activation of different antioxidant pathways. It is concluded that the effects of GCs are variable, possibly due to the difference between the affinity and inhibition of the NKA pump.

LOCALIZAÇÃO DA Na+, K+-ATPase EM ESPERMATOZOIDES OVINO IN NATURA, PÓS-DESCONGELAÇÃO E CAPACITADOS IN VITRO E DETERMINAÇÃO DOS EFEITOS DE SEU BLOQUEIO SOBRE AS CARACTERÍSTICAS ESPERMÁTICAS GARANHUNS 2018

Objetivando-se localizar a Na+, K+-ATPase em espermatozoides ovinos (in natura, pós decongelação e capacitados in vitro), assim como avaliar a influência da adição da ouabaína sobre os parâmetros cinemáticos espermáticos, foram realizados dois experimentos. Exp.1: amostras de sêmen (quatro carneiros, sete repetições) foram colhidas, diluídas (Trisgema, concentração final de 200 x 106 espermatozoides/mL) e congeladas à -196 C°. Exp. 2: amostras seminais de quatro ovinos (cinco repetições) foram diluídas em meio TALPm (50 x106 espermatozoides/mL) e aliquotadas em dois grupos: parte destinada a capacitação in vitro (adição de 2,5% de soro de ovelha em estro - SOE) ou com adição de ouabaína (10-4 M), visando o bloqueio da Na+,K+-ATPase. Alíquotas de sêmen de ambos os experimentos foram avaliadas pelo sistema CASA para análise dos parâmetros de cinemética espermática, e por meio da citometria de fluxo, para a localização da Na+, K+-ATPase (Bodipy FL Ouabaína), análise da integridade de membrana plasmática (IP) e acrossomal (FITC-PNA), viabilidade e estabilidade de membrana plasmática (YO-PRO e M540). No Exp. 2, além das análises supracitadas realizou-se também a avaliação do potencial de membrana mitocondrial (JC-1). Para o Exp. 2, todas as análises foram efetudas após 0h e 3h de incubação a 36 °C. Os resultados obtidos foram: 1) a Na+, K+-ATPase foi localizada na peça intermediária dos espermatozoides independente do status da amostra (in natura, pós descongelação e capacitados); 2) o processo de congelação/descongelação resultou em uma diminuição (P<0,05) nos parâmetros de MT, MP e VAP, um aumento no BCF, além de redução (P<0,05) no percentual de células apresentando marcação com Bodipy FL Ouabaína, com membranas plasmáticas e acrossomal íntegras, e percentagem de células com membrana plasmática estabilizada; 3) A longo do período de incubação, o bloqueio da Na+, K+-ATPase resultou na redução (P<0,05) da MP, VCL e VSL, enquanto no grupo controle observou-se redução (P <0,05) dos parâmetros MT, VCL e ALH; 4) A adição do SOE ao meio de TALPm resultou em redução (P<0,05) em MP, VCL e ALH, enquanto no grupo controle reduziu (P<0,05) os valores de MT e MP, não havendo diferença entre tratamentos para os parâmentros cinemáticos. Além disso, independentemente dos tratamentos, observou-se ao longo da incubação elevação (P<0,05) no percentual de células apresentando membrana plasmática lesionadas, acrossomal íntegra sem marcação da Na+, K+-ATPase. Em conclusão, independente do status fisiológico, a Na+, K+-ATPase não alterou sua localização, permanecendo na peça intermediária dos espermatozoides ovinos, e que, sua inibição não influenciou sobre os parâmetros de motilidade espermática.

Aiming to localize the Na+, K+-ATPase in ram sperm (raw, post-thaw and capacitated in vitro), and to evaluate the influience of ouabain addition on kinematic sperm parameters, two experiments were performed. Exp.1: semen samples (four rams, seven replicates) were harvested, diluted (Tris-gema, final concentration 200 x 106 sperm/mL) and frozen at -196 C°. Exp. 2: sêmen samples from four rams (five replicates) were diluted in TALPm (50 x106 sperm/mL) and aliquot for two precedures: in vitro capacitation (addition of 2,5% of estrous sheep serum - ESS) or ouabain addition (10-4 M), in order to block the Na+,K+- ATPase. Semen aliquots from both experiments were evaluated using CASA system for kinematic parameters and with aid of flow cytometry it was determined localization of Na+, K+-ATPase (Bodipy FL Ouabaína), evaluated plasmatic (IP) and acrosomal membrande (FITC-PNA) integrity, viability and stability of sperm plasma membrane (YO-PRO e M540). During Exp. 2, it was also evaluated the mitochondrial membrane potential (JC-1). For Exp. 2, all analysis were performed at 0h and 3h of incubation time (36 °C). These are the results: 1) The Na+, K+-ATPase was localized on middle piece of sperm flagellum, despite sample status (raw, post-thaw and capacitated); 2) the freezing-thawing proccess reduced (P<0,05) MT, MP and VAP and increased BCF. There was also a reduction (P<0.05) on percentage of sperm stained with Bodipy FL Ouabain, with intact plasmatic and acrossomal membranes, and percentage of sperm with stabilized plasma membrane; 3) Throught incubation time, the Na+, K+-ATPase blocking reduced (P<0.05) MP, VCL and VSL, whereas on control group reduced (P <0.05) MT, VCL and ALH; 4) The addition of ESS to TALPm reduced (P<0.05) MP, VCL and ALH, and on control group reduced (P<0.05) MT and MP, with no difference between treatments for kinematic parameters. Therefore, despite of treatment, there was an increasing (P<0.05) on percentage of sperm with damages plasma membrane, intact acrosome and no stain for Na+, K+-ATPase. In conclusion, despit of sperm phisiological status, Na+, K+- ATPase ramains on middle piece of ram sperm flagellum and its inhibition have no influence on sperm motility parameters.