Muscarinic and nicotinic ACh receptor activation differentially mobilize Ca2+ in rat intracardiac ganglion neurons

The origin of intracellular Ca2+ concentration ([Ca2+](i)) transients stimulated by nicotinic ( nAChR) and muscarinic ( mAChR) receptor activation was investigated in fura-2-loaded neonatal rat intracardiac neurons. ACh evoked [Ca2+](i) increases that were reduced to similar to 60% of control in the presence of either atropine ( 1 muM) or mecamylamine ( 3 muM) and to < 20% in the presence of both antagonists. Removal of external Ca2+ reduced ACh-induced responses to 58% of control, which was unchanged in the presence of mecamylamine but reduced to 5% of control by atropine. The nAChR-induced [Ca2+](i) response was reduced to 50% by 10 ��M ryanodine, whereas the mAChR-induced response was unaffected by ryanodine, suggesting that Ca2+ release from ryanodine-sensitive Ca2+ stores may only contribute to the nAChR-induced [Ca2+](i) responses. Perforated-patch whole cell recording at - 60 mV shows that the rise in [Ca2+](i) is concomitant with slow outward currents on mAChR activation and with rapid inward currents after nAChR activation. In conclusion, different signaling pathways mediate the rise in [Ca2+](i) and membrane currents evoked by ACh binding to nicotinic and muscarinic receptors in rat intracardiac neurons.

A low concentration of ouabain (0.18 ��g/kg) enhances hypertension in spontaneously hypertensive rats by inhibiting the Na+ pump and activating the renin-angiotensin system

We investigated the effects of low ouabain concentrations on systolic (SAP) and diastolic (DAP) arterial pressures and on pressor reactivity in 3-month-old male spontaneously hypertensive rats (SHR). Arterial blood pressure (BP) and pressor reactivity to phenylephrine (PHE) were investigated before and after 0.18 ��g/kg ouabain administration (N = 6). The influence of hexamethonium (N = 6), canrenone (N = 6), enalapril (N = 6), and losartan (N = 6) on ouabain actions was evaluated. Ouabain increased BP (SAP: 137 �� 5.1 to 150 �� 4.7; DAP: 93.7 �� 7.7 to 116 �� 3.5 mmHg; P < 0.05) but did not change PHE pressor reactivity. Hexamethonium reduced basal BP in control but not in ouabain-treated rats. However, hexamethonium + ouabain increased DAP sensitivity to PHE. Canrenone did not affect basal BP but blocked ouabain effects on SAP. However, after canrenone + ouabain administration, DAP pressor reactivity to PHE still increased. Enalapril and losartan reduced BP and abolished SAP and DAP responses to ouabain. Enalapril + ouabain reduced DAP reactivity to PHE, while losartan + ouabain reduced SAP and DAP reactivity to PHE. In conclusion, a small dose of ouabain administered to SHR increased BP without altering PHE pressor reactivity. Although the renin-angiotensin system (RAS), Na+ pump and autonomic reflexes are involved in the effects of ouabain on PHE reactivity, central mechanisms might blunt the actions of ouabain on PHE pressor reactivity. The effect of ouabain on SAP seems to depend on the inhibition of both Na+ pump and RAS, whereas the effect on DAP seems to depend only on RAS.

Effects of ouabain on vascular reactivity

Ouabain is an endogenous substance occurring in the plasma in the nanomolar range, that has been proposed to increase vascular resistance and induce hypertension. This substance acts on the a-subunit of Na+,K+-ATPase inhibiting the Na+-pump activity. In the vascular smooth muscle this effect leads to intracellular Na+ accumulation that reduces the activity of the Na+/Ca2+ exchanger and to an increased vascular tone. It was also suggested that circulating ouabain, even in the nanomolar range, sensitizes the vascular smooth muscle to vasopressor substances. We tested the latter hypothesis by studying the effects of ouabain in the micromolar and nanomolar range on phenylephrine (PE)-evoked pressor responses. The experiments were performed in normotensive and hypertensive rats in vivo, under anesthesia, and in perfused rat tail vascular beds. The results showed that ouabain pretreatment increased the vasopressor responses to PE in vitro and in vivo. This sensitization after ouabain treatment was also observed in hypertensive animals which presented an enhanced vasopressor response to PE in comparison to normotensive animals. It is suggested that ouabain at nanomolar concentrations can sensitize vascular smooth muscle to vasopressor stimuli possibly contributing to increased tone in hypertension.

Histamine induces ATP release from human subcutaneous fibroblasts via pannexin-1 hemichannels leading to Ca2+ mobilization and cell proliferation

Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca2+ ([Ca2+]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca2+]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca2+]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with 10Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca2+]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADPsensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca2+]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors.

Bradykinin-induced Ca2+ signaling in human subcutaneous fibroblasts involves ATP release via hemichannels leading to P2Y12 receptors activation

Background: Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts. Results: Bradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2- octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor. Conclusions: Bradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors.

Digitally programmable delay-locked-loop with adaptive charge pump current for UWB radar system

Disserta����o apresentada na Faculdade de Ci��ncias e Tecnologia da Universidade Nova de Lisboa para obten����o do grau de Mestre em Engenharia Electrot��cnica e Computadores

Biological activity of well defined hydantoin derivatives on efflux pump systems of bacteria and cancer cells

A multi-resist��ncia a antibi��ticos e medicamentos usados em quimioterapia �� um dos grandes problemas com os quais as institui����es de sa��de se debatem hoje em dia. A ac����o provocada por bombas de efluxo �� uma das suas causas. Estas bombas t��m uma import��ncia fundamental, uma vez que, ao expelirem todo o tipo de t��xicos para o exterior das c��lulas, tamb��m expelem medicamentos, fazendo com que estes n��o tenham o efeito desejado dentro delas. As bombas de efluxo s��o transportadores que se encontram nas membranas de todo o tipo de c��lulas. Existem dois grandes tipos de bombas de efluxo: as prim��rias e as secund��rias. As primeiras conferem multi-resist��ncia principalmente em c��lulas eucariotas, como as c��lulas do cancro em humanos, tendo como fun����o a media����o da repulsa de subst��ncias t��xicas por interm��dio da hidr��lise de ATP. A primeira a ser descoberta e mais estudada destas bombas foi a ABCB1 que �� o gene que codifica a glicoprote��na-P (P de permeabilidade). Enquanto as secund��rias, que s��o a maior fonte de multi-resist��ncia em bact��rias, promovem a extrus��o de subst��ncias t��xicas atrav��s da for��a motriz de prot��es. Neste tipo de bombas s��o conhecidas quatro fam��lias principais, das quais uma das mais importantes �� a superfam��lia RND, uma vez que inclui a bomba AcrAB-TolC, que �� muito importante no metabolismo xenobi��tico de bact��rias Gramnegativas, nomeadamente a E.coli. Com o objectivo de reverter a multi-resist��ncia, tanto em c��lulas eucariotas como procariotas, t��m-se desenvolvido estrat��gias de combate que envolvem a descoberta de subst��ncias que inibam as bombas de efluxo. Assim sendo, ao longo dos tempos t��m sido descobertas variadas subst��ncias que cumprem este objectivo. �� o caso, por exemplo, dos derivados de fluoroquinolonas usados como inibidores de bombas de efluxo em bact��rias ou do Tamoxifen, utilizado na terapia de pacientes com cancro da mama. Um dos grupos de subst��ncias estudados para o desenvolvimento de poss��veis compostos que actuem como reversores de multi-resist��ncia s��o os compostos derivados de hidanto��nas. Estes, s��o conhecidos por possu��rem uma grande variedade de propriedades bioqu��micas e farmacol��gicas, sendo portanto usados para tratarem algumas doen��as em humanos, como a epilepsia. Nestes, est��o englobados compostos com actividade anti-convuls��o que constitui a sua grande mais-valia e, dependente da substitui����o no anel que os constitui, uma grande variedade de outras propriedades farmacol��gicas como a anti-fungica, a anti-arritmica, a anti-viral, a anti-diab��tica ou por exemplo a antagoniza����o de determinados receptores, como os da serotonina. Apesar de pouco usados em estudos experimentais para desenvolver subst��ncias anti-carcinog��nicas, existem alguns estudos com este efeito. Objectivos: O presente projecto envolve o estudo de bombas de efluxo prim��rias e secund��rias, em c��lulas eucariotas e procariotas, respectivamente. Em bact��rias, foram usados quatro modelos experimentais: Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, E. coli AG 100 e Salmonella Enteritidis NCTC 13349. Em c��lulas de cancro foram usadas, c��lulas T de linfoma de rato parentais e c��lulas T de linfoma de rato transfectadas com o gene humano MDR-1. O principal objectivo deste estudo foi a pesquisa de novos moduladores de bombas de efluxo presentes em bact��rias e c��lulas do cancro, tentando assim contribuir para o desenvolvimento de novos agentes farmacol��gicos que consigam reverter a multi-resist��ncia a medicamentos. Assim sendo foram testados trinta compostos derivados de hidanto��nas: SZ-2, SZ-7, LL-9, BS-1, JH-63, MN-3, TD-7k, GG-5k, P3, P7, P10, P11, RW-15b, AD-26, RW-13, AD-29, KF-2, PDPH-3, Mor-1, KK-XV, Thioam-1, JHF-1, JHC-2, JHP-1, Fur-2, GL-1, GL-7, GL-14, GL-16, GL-18. Como forma de atingir estes objectivos, a actividade biol��gica dos trinta compostos derivados de hidanto��nas foi avaliada nas quatro estirpes de bact��rias da seguinte forma: foram determinadas as concentra����es m��nimas inibit��rias dos trinta compostos como forma de definir as concentra����es em que os compostos seriam utilizados. Os compostos foram posteriormente testadas com um m��todo fluorom��trico de acumula����o de brometo de et��deo, que �� um substrato comum em bombas de efluxo bacterianas, desenvolvido por Viveiros et al. A actividade biol��gica dos compostos derivados de hidanto��nas nas c��lulas de cancro foi demonstrada por diferentes m��todos. O efeito anti-proliferativo e citot��xico dos trinta compostos foi avaliado nas c��lulas T de linfoma de rato transfectadas com o gene humano MDR-1 pelo m��todo de thiazolyl de tetraz��lio (MTT). Como o brometo de et��deo tamb��m �� expelido pelos transportadores ABC, estes compostos foram posteriormente testados com um m��todo fluorom��trico de acumula����o de brometo de et��deo desenvolvido por Spengler et al nos dois diferentes tipos de c��lulas eucariotas. Resultados: A maioria dos compostos derivados de hidanto��nas foi eficaz na modula����o de bombas de efluxo, nas duas estirpes de bact��rias Gram-negativas e nos dois diferentes tipos de c��lulas T de linfoma. Em contraste com estes resultados, nas duas estirpes de c��lulas Gram-positivas, a maioria dos compostos tiveram pouco efeito na inibi����o de bombas de efluxo ou at�� nenhum, em muitos dos casos. De uma maneira geral os melhores compostos nas diferentes estirpes de bact��rias foram: Thioam-1, SZ-2, P3, Rw-15b, AD-26, AD-29, GL-18, GL-7, KF-2, SZ-7, MN-3, GL-16 e GL- 14. Foram portanto estes os compostos que provocaram maior acumula����o de brometo de et��deo, inibindo assim com maior efic��cia as bombas de efluxo. No presente estudo, a maioria dos compostos conseguiu inibir a resist��ncia provocada pela bomba de efluxo ABCB1, tanto nas c��lulas parentais bem como nas c��lulas que sobre-expressam esta bomba, causando a acumula����o de brometo de et��deo dentro das c��lulas. As c��lulas que sobreexpressam a bomba ABCB1 foram posteriormente testadas com citometria de fluxo que �� a t��cnica padr��o para pesquisa de inibidores de bombas de efluxo. Os compostos que foram mais efectivos na inibi����o da bomba ABCB1, causando assim maior acumula����o de brometo de et��deo nas c��lulas que sobre-expressam esta bomba foram: PDPH-3, GL-7, KK-XV, AD-29, Thioam-1, SZ-7, KF-2, MN-3, RW-13, LL-9, P3, AD-26, JH-63 e RW- 15b. Este facto n��o corroborou totalmente os resultados da citometria de fluxo uma vez que os moduladores que provocaram maior inibi����o da bomba ABCB1 foram o MN-3, JH-63 e o BS-1, sendo que o ��ltimo n��o foi seleccionado como um bom composto usando o m��todo fluorom��trico de acumula����o de brometo de et��deo. Conclus��o: Os compostos derivados de hidanto��nas testados tiveram maior efeito nas estirpes de bact��rias Gram-negativas do que nas Gram-positivas. Relativamente ��s c��lulas eucariotas, as estruturas mais activas apresentam substituintes arom��ticos bem como alguns fragmentos aminicos terci��rios.

The inhibitory activity of synthetic compounds and ions against transporters of multi-drug resistant bacteria

RESUMO: Sessenta e tr��s derivados de hidanto��na foram utilizados para avaliar poss��veis efeitos de modula����o na actividade das bombas de efluxo (BE) na Salmonella NCTC 13349 utilizando um m��todo fluorim��trico semi-autom��tico. Nenhum dos compostos apresentaram actividade anti-bacteriana at�� concentra����es de 240 mg/L. Entre todos os compostos, SZ-7 demonstrou possuir propriedades de modula����o de effluxo na presen��a de glucose. Para testar esta actividade, estirpes de Salmonella resistentes �� ciprofloxacina, induzidas a elevados n��veis de resist��ncia com sobre-express��o de BE, foram expostas ao SZ-7. Este derivado afectou a susceptibilidade das estirpes �� ciprofloxacina. Uma vez que os 63 compostos estudados apresentaram pouco efeito inibit��rio /cumulativo, apesar de serem conhecidos pelos seus efeitos moduladores de BE-dependentes de i��es em eucariotas, foi questionado o papel dos i��es na regula����o de BE bacterianas, que poder��o influenciar a efic��cia de novos compostos. Para este estudo, utilizamos a Escherichia coli AG100 como modelo, devido ao extenso conhecimento no que respeita a estrutura e actividade das BE. Devido �� import��ncia de i��es de c��lcio (Ca2+) nos canais de transporte membranar e na actividade de ATPases, a sua actividade na modula����o do efluxo foi investigada. De resultados anteriormente obtidos concluiu-se que a pH 5 o efluxo �� independente de energia metab��lica; contudo, a pH 8 �� absolutamente dependente, sendo que o Ca2+ �� indispens��vel para manter a actividade das ATPases bacterianas. A acumula����o/effluxo de EtBr pela E. coli AG100 foi determinada na presen��a/aus��ncia de Ca2+, clorpromazina (inibidor de liga����o de Ca2+ a prote��nas), e ��cido etilenodiamino tetra-ac��tico (quelante de Ca2+). Acumula����o/effluxo aumentou a pH 8, contudo o Ca2+ reverte estes efeitos evidenciando a sua import��ncia no funcionamento das BE bacterianas. Em resumo este trabalho colocou em evid��ncia que muitos aspectos bioqu��micos e bioenerg��ticos devem ser tomados em considera����o no estudo da resist��ncia bacteriana mediada por BE.------- ABSTRACT: Sixty-three hydantoin derivatives were evaluated for their modulating effects on efflux pump (EP) activity of Salmonella NCTC 13349 utilizing a semi-automatic fluorometric method. None of the compounds presented antibacterial activities at concentrations as high as 240 mg/L. Among all compounds, SZ-7 showed possible efflux modulating activity in the presence of glucose, indicative of a potential EP inhibitor. To verify its potential effects, ciprofloxacin-resistant Salmonella strains, induced to high level resistance with over-expressing EPs, were exposed to SZ-7. This derivative affected the susceptibility of the ciprofloxacin-resistant strains. Since the 63 compounds studied had very low inhibitory/accumulative effects, even though their known for being efficient in modulating ion-driven eukaryotic EPs, we questioned whether ions had a leading role in regulating bacterial EPs, influencing the effectiveness of new compounds. For this study we used Escherichia coli AG100 as a model, due to the extensive knowledge on its EPs structure and activity. Owing the importance of calcium ions (Ca2+) for membrane transport channels and activity of ATPases, the role of Ca2+ was investigated. From previous results we concluded that at pH 5 efflux is independent of metabolic energy; however, at pH 8 it is entirely dependent of metabolic energy and the Ca2+ ions are essential to maintain the activity of bacterial ATPases. Accumulation and efflux of ethidium bromide (EtBr) by E. coli AG100 was determined in the presence and absence of Ca2+, chlorpromazine (inhibitor of Ca2+-binding to proteins), and ethylenediaminetetraacetic acid (Ca2+ chelator). Accumulation of EtBr increased at pH 8; however Ca2+ reversed these effects providing information as to the importance of this ion in the regulation of bacterial EP systems. Overall this work puts in evidence that many biochemical and bioenergetic aspects related to the strains physiology need to be taken into consideration in bacterial drug resistance mediated by EPs.

Factors affecting Helicobacter pylori eradication using a seven-day triple therapy with a proton pump inhibitor, tinidazole and clarithromycin, in brazilian patients with peptic ulcer

Triple therapy is accepted as the treatment of choice for H. pylori eradication. In industrialized countries, a proton pump inhibitor plus clarithromycin and amoxicillin or nitroimidazole have shown the best results. Our aims were: 1. To study the eradication rate of the association of a proton pump inhibitor plus tinidazole and clarithromycin on H. pylori infection in our population. 2. To determine if previous treatments, gender, age, tobacco, alcohol use, and non-steroidal anti-inflammatory drugs (NSAIDs) change the response to therapy. METHODS: Two hundred patients with peptic ulcer (upper endoscopy) and H. pylori infection (histology and rapid urease test - RUT) were included. A proton pump inhibitor (lansoprazole 30 mg or omeprazole 20 mg), tinidazole 500 mg, and clarithromycin 250 mg were dispensed twice a day for a seven-day period. Eradication was assessed after 10 to 12 weeks of treatment through histology and RUT. RESULTS: The eradication rate of H. pylori per protocol was 65% (128/196 patients). This rate was 53% for previously treated patients, rising to 76% for not previously treated patients, with a statistical difference p<0.01. No significant difference was observed regarding sex, tobacco use, alcohol consumption, and NSAID use, but for elderly patients the difference was p = 0.05. Adherence to treatment was good, and side effects were mild. CONCLUSIONS: A proton pump inhibitor, tinidazole, and clarithromycin bid for seven days resulted in H. pylori eradication in 65% of the patients. Previous treatments were the main cause of treatment failure.

Intraaortic balloon pump support during coronary angioplasty. Initial experience

OBJECTIVE: To evaluate the use of the intraaortic balloon (IAoB) in association with coronary angioplasty in high-risk patients. METHODS: Fourteen high-risk patients unresponsive to clinical therapy and with formal contraindication to surgical revascularization were treated by coronary angioplasty, most of which was followed by stenting. All procedures were performed with circulatory support with the IAoB. This study reports the early results and the late findings after 12 months of follow-up. Six patients had multivessel coronary disease; of these, four had left main equivalent lesions and two had unprotected left main coronary artery disease, one of whom had severe "end-vessel" stenosis and the other was a patient with Chagas' disease with single-vessel lesion. Eleven patients had a left ventricular ejection fraction <30%. RESULTS: In 100% of the patients, the procedures were initially successful. Two patients had severe bleeding during the withdrawal of the left femoral sheath. At the end of twelve months, 4 patients were asymptomatic and the others were clinically controlled. There were two late deaths in the 7th and 11th months. CONCLUSION: The combined use of the intraaortic balloon pump and percutaneous coronary angioplasty in high-risk patients with acute ischemic syndromes provides the necessary hemodynamic stability to successfully perform the procedures.

Importancia del Ca2+ en la fisiolog��a esperm��tica: modulaci��n de su actividad por progesterona y prote��nas del plasma seminal. Importance of calcium in sperm physiology: its regulation by progesterone and proteins from the seminal plasma.

El objetivo general del proyecto es estudiar el efecto de la progesterona y de algunas prote��nas del plasma seminal sobre la actividad del Ca2+ en diferentes procesos fisiol��gicos que ocurren en el espermatozoide, los cuales est��n estrechamente relacionados con la capacidad fertilizante de esta c��lula. La progesterona, principal esteroide secretado por las c��lulas del cumulus oophorus, ejerce su efecto a trav��s de un receptor no-gen��mico provocando aumento en el calcio intracelular de los espermatozoides y, consecuentemente, promoviendo la capacitaci��n, la respuesta quimiot��ctica y la exocitosis acrosomal. Pese a estas observaciones, los mecanismos a trav��s de los cuales la progesterona estimula fen��menos tan diversos en el espermatozoide son a��n desconocidos. Tampoco se conoce con exactitud el papel funcional y los mecanismos de acci��n de algunas prote��nas del plasma seminal que interaccionan y se unen a los espermatozoides, con alta especificidad, durante la eyaculaci��n. Por lo tanto, resulta altamente interesante profundizar los estudios sobre las propiedades funcionales de las prote��nas caltrin (calcium transport inhibitor) y ��-microseminoprotein (MSP) del plasma seminal de mam��feros, las cuales responden a las caracter��sticas mencionadas. Los estudios hasta ahora realizados han dado cuenta de que caltrin inhibe la incorporaci��n de Ca2+ extracelular, previene la exocitosis acrosomal espont��nea y promueve la uni��n espermatozoide-zona pel��cida. Tambi��n hay datos preliminares que sugieren un efecto inhibitorio sobre la movilidad hiperactivada de los espermatozoides. Respecto a MSP, s��lo se sabe que inhibe la exocitosis acrosomal espont��nea y que su contenido, en el plasma seminal, guarda una relaci��n inversa con la fertilidad. Por todo lo expuesto, se propone estudiar los mecanismos de acci��n de la progesterona y las prote��nas caltrin y MSP sobre los procesos fisiol��gicos antes indicados. Para ello, se estudiar��n las variaciones de Ca2+ intracelular en espermatozoides individuales sometidos a diferentes tratamientos (gradientes de progesterona, capacitaci��n en presencia y ausencia de caltrin y/o MSP, etc.), usando video microscop��a de fluorescencia y an��lisis computarizado de im��genes. Tambi��n se examinar�� la influencia de estas mol��culas sobre la interacci��n de gametas y la fertilizaci��n.

Mecanismo de interacci��n, calcio dependiente, entre Calcineurina y PERK, involucrado en el Estr��s de Ret��culo Endopl��smico. Ca2+ -dependent mechanism of interaction between Calcineurin and PERK involved in Endoplasmic Reticulum Stress.

El Estr��s de Ret��culo Endopl��smico (RE) es inducido por la acumulaci��n de prote��nas sin plegar en el lumen de la organela. Esto se puede observar en diversas situaciones fisio-patol��gicas como durante una infecci��n viral o en proceso isqu��mico. Adem��s, contribuye a la base molecular de numerosas enfermedades ya sea ��ndole metab��lico (Fibrosis qu��stica o Diabetes Miellitus) o neurodegenerativas como mal de Alzheimer o Parkinson (Mutat Res, 2005, 569). Para restablecer la homeostasis en la organela se activa una se��al de transducci��n (UPR), cuya respuesta inmediata es la atenuaci��n de la s��ntesis de prote��na debido a la fosforilaci��n de subunidad alpha del factor eucari��tico de iniciaci��n de translaci��n (eIF2��) v��a PERK. Esta es una prote��na de membrana de RE que detecta estr��s. Bajo condiciones normales, PERK est�� inactiva debido a la asociaci��n de su dominio luminar con la chaperona BIP (Nat Cell Biol, 2000, 2: 326). Frente a una situaci��n de estr��s, la chaperona se disocia causando desinhibici��n. Recientemente, (Plos One 5: e11925) se observ��, bajo condiciones de estr��s, un aumento de Ca2+ citos��lico y un r��pido incremento de la expresi��n de calcineurina (CN), una fosfatasa citos��lica dependiente de calcio, heterodim��rica formada por una subunidad catal��tica (CN-A) y una regulatoria (CN-B). Adem��s, CN interacciona, sin intermediarios, con el dominio citos��lico de PERK favoreciendo su trans-autofosforilaci��n. Resultados preliminares indican que, astrocitos CNA��-/- exhibieron, en condiciones basales, un mayor n��mero de c��lulas muertas y de niveles de eIF2�� fosforilado que los astrocitos CNA��-/-. Hip��tesis: CNA��/B interacciona con PERK cuando el Ca2+ citos��lico esta incrementado luego de haberse inducido Estr��s de RE, lo cual promueve dimerizaci��n y auto-fosforilaci��n de la quinasa, acentu��ndose as�� la fosforilaci��n de eIF2�� e inhibici��n de la s��ntesis de prote��nas. Esta activaci��n citos��lica de PERK colaborar��a con la ya descrita, desinhibici��n luminal llevada cabo por BIP. Cuando el Ca2+ citos��lico retorna a los niveles basales, PERK fosforila a CN, reduciendo su afinidad de uni��n y disoci��ndose el complejo CN/PERK. Objetivo general: Definir las condiciones por las cuales CN interacciona con PERK y regula la fosforilaci��n de eIF2�� e inhibici��n de la s��ntesis de prote��na. Objetivos espec��ficos: I-Estudiar la diferencia de afinidades y dependencia de Ca2+, de las dos isoformas de CN (�� y ��) en su asociaci��n con PERK. Adem��s verificar la posible participaci��n de la subunidad B de CN en esta interacci��n. II-Determinar si la auto-fosforilaci��n de PERK es diferencialmente regulada por las dos isoformas de CN. III-Discernir la relaci��n del estado de fosforilaci��n de CN con su uni��n a PERK. IV-Determinar efectos fisiol��gicos de la interacci��n de CN-PERK durante la respuesta de Estr��s de RE. Para llevar a cabo este proyecto se realizar��n experimentos de biolog��a molecular, interacci��n prote��na-prote��na, ensayos de fosforilaci��n in vitro y un perfil de polisoma con astrocitos CNA��-/- , CNA���-/- y astrocitos controles. Se espera encontrar una mayor afinidad de uni��n a PERK de la isoforma �� de CN y en condiciones donde la concentraci��n de Ca2+ sea del orden micromolar e imite niveles del i��n durante un estr��s. Con respecto al estado de fosforilaci��n de CN, debido a los resultados preliminares, donde solo se la encontr�� fosforilada en condiciones basales, se piensa que CN podr��a interactuar con mayor afinidad con PERK cuando CN se encuentre desfosforilada. Por ��ltimo, se espera encontrar un aumento de eIF2�� fosforilado y una acentuaci��n de la atenuaci��n de la s��ntesis de prote��na como consecuencia de la mayor activaci��n de PERK por su asociaci��n con la isoforma �� de CN en astrocitos donde el Estr��s de RE se indujo por privaci��n de oxigeno y glucosa. Estos experimentos permitir��n avanzar en el estudio de una nueva funci��n citoprotectora de CN recientemente descrita por nuestro grupo de trabajo y sus implicancias en un modelo de isquemia. The accumulation of unfolded proteins into the Endoplasmic Reticulum (ER) activates a signal transduction cascade called Unfolding Protein Response (UPR), which attempts to restore homeostasis in the organelle. (PKR)-like-ER kinase (PERK) is an early stress response transmembrane protein that is generally inactive due to its association with the chaperone BIP. During ER stress, BIP is tritrated by the unfolded protein, leading PERK activation and phosphorylation of eukaryotic initiation factor-2 alpha (eIF2alpha), which attenuates protein s��ntesis. If ER damage is too great and homeostasis is not restored within a certain period of time, an apoptotic response is elicited. We recently demonstrated a cytosolic Ca2+ increase in Xenopus oocytes after induce ER stress. Moreover, calcineurin A/B, a an heterotrimeric Ca2+ dependent phosphatases (CN-A/B), associates with PERK increasing its auto-phosphorylation and significantly enhancing cell viability. Preliminary results suggest that, CN-A��-/- knockout astrocytes exhibit a significant higher eIF2�� phosphorylated level compared to CN-A��-/- astrocytes. Our working hypothesis establishes that: CN binds to PERK when cytosolic Ca2+ is initially increased by ER stress, promoting dimerization and autophosphorylation, which leads to phosphorylation of elF2�� and subsequently attenuation of protein translation. When cytosolic Ca2+ returns to resting levels, PERK phosphorylates CN, reducing its binding affinity so that the CN/PERK complex dissociates. The goal of this project is to determine the conditions by which CN binding to PERK attenuates protein translation during the ER stress response and subsequently, to determine how the interaction of CN with PERK is terminated when stress is removed. To perform this project is planed to do molecular biology experiments, pull down assays, in vitro phosphorylations and assess overall mRNA translation efficiency doing a polisome profile.

Exerc��cio e restri����o alimentar aumentam o RNAm de prote��nas do tr��nsito de Ca2+ mioc��rdico em ratos

FUNDAMENTO: Treinamento f��sico (TF) aumenta a sensibilidade dos horm��nios tireoidianos (HT) e a express��o g��nica de estruturas moleculares envolvidas no movimento intracelular de c��lcio do mioc��rdio, enquanto a restri����o alimentar (RIA) promove efeitos contr��rios ao TF. OBJETIVO: Avaliar os efeitos da associa����o TF e RIA sobre os n��veis plasm��ticos dos HT e a produ����o de mRNA dos receptores HT e estruturas moleculares do movimento de c��lcio do mioc��rdio de ratos. M��TODOS: Utilizaram-se ratos Wistar Kyoto divididos em: controle (C, n = 7), RIA (R50, n = 7), exerc��cio f��sico (EX, n = 7) e exerc��cio f��sico + RIA (EX50, n = 7). A RIA foi de 50% e o TF foi nata����o (1 hora/dia, cinco sess��es/semana, 12 semanas consecutivas). Avaliaram-se as concentra����es s��ricas de triiodotironina (T3), tiroxina (T4) e horm��nio tireotr��fico (TSH). O mRNA da bomba de c��lcio do ret��culo sarcoplasm��tico (SERCA2a), fosfolamban (PLB), trocador Na+/Ca+2 (NCX), canal lento de c��lcio (canal-L), rianodina (RYR), calsequestrina (CQS) e receptor de HT (TR&#945;1 e TR&#946;1) do mioc��rdio foram avaliados por rea����o em cadeia da polimerase (PCR) em tempo real. RESULTADOS: RIA reduziu o T4, TSH e mRNA do TR&#945;1 e aumentou a express��o da PLB, NCX e canal-L. TF aumentou a express��o do TR&#946;1, canal-L e NCX. A associa����o TF e RIA reduziu T4 e TSH e aumentou o mRNA do TR&#946;1, SERCA2a, NCX, PLB e correla����o do TR&#946;1 com a CQS e NCX. CONCLUS��O: Associa����o TF e RIA aumentou o mRNA das estruturas moleculares c��lcio transiente, por��m o eixo HT-receptor n��o parece participar da transcri����o g��nica dessas estruturas.

Chronic Stress Improves NO- and Ca2+ Flux-Dependent Vascular Function: A Pharmacological Study

Background: Stress is associated with cardiovascular diseases. Objective: This study aimed at assessing whether chronic stress induces vascular alterations, and whether these modulations are nitric oxide (NO) and Ca2+ dependent. Methods: Wistar rats, 30 days of age, were separated into 2 groups: control (C) and Stress (St). Chronic stress consisted of immobilization for 1 hour/day, 5 days/week, 15 weeks. Systolic blood pressure was assessed. Vascular studies on aortic rings were performed. Concentration-effect curves were built for noradrenaline, in the presence of L-NAME or prazosin, acetylcholine, sodium nitroprusside and KCl. In addition, Ca2+ flux was also evaluated. Results: Chronic stress induced hypertension, decreased the vascular response to KCl and to noradrenaline, and increased the vascular response to acetylcholine. L-NAME blunted the difference observed in noradrenaline curves. Furthermore, contractile response to Ca2+ was decreased in the aorta of stressed rats. Conclusion: Our data suggest that the vascular response to chronic stress is an adaptation to its deleterious effects, such as hypertension. In addition, this adaptation is NO- and Ca2+-dependent. These data help to clarify the contribution of stress to cardiovascular abnormalities. However, further studies are necessary to better elucidate the mechanisms involved in the cardiovascular dysfunction associated with stressors. (Arq Bras Cardiol. 2014; [online].ahead print, PP.0-0)

Obesity Resistance Promotes Mild Contractile Dysfunction Associated with Intracellular Ca2+ Handling

Abstract Background: Diet-induced obesity is frequently used to demonstrate cardiac dysfunction. However, some rats, like humans, are susceptible to developing an obesity phenotype, whereas others are resistant to that. Objective: To evaluate the association between obesity resistance and cardiac function, and the impact of obesity resistance on calcium handling. Methods: Thirty-day-old male Wistar rats were distributed into two groups, each with 54 animals: control (C; standard diet) and obese (four palatable high-fat diets) for 15 weeks. After the experimental protocol, rats consuming the high-fat diets were classified according to the adiposity index and subdivided into obesity-prone (OP) and obesity-resistant (OR). Nutritional profile, comorbidities, and cardiac remodeling were evaluated. Cardiac function was assessed by papillary muscle evaluation at baseline and after inotropic maneuvers. Results: The high-fat diets promoted increase in body fat and adiposity index in OP rats compared with C and OR rats. Glucose, lipid, and blood pressure profiles remained unchanged in OR rats. In addition, the total heart weight and the weight of the left and right ventricles in OR rats were lower than those in OP rats, but similar to those in C rats. Baseline cardiac muscle data were similar in all rats, but myocardial responsiveness to a post-rest contraction stimulus was compromised in OP and OR rats compared with C rats. Conclusion: Obesity resistance promoted specific changes in the contraction phase without changes in the relaxation phase. This mild abnormality may be related to intracellular Ca2+ handling.