Envelhecimento e alterações cardíacas, bioquímica, moleculares e funcionais: estudo experimental / Aging and Cardiac, Biochemical, Molecular and functional changes: an experimental study

Fundamentos: O envelhecimento abrange mudanças físicas e psicológicas que reduzem a capacidade de adaptação do idoso à sociedade, sendo o maior fator de risco para doenças cardiovasculares. Objetivo: Investigar alterações do sistema cardiovascular decorrentes do processo de envelhecimento em ratos. Métodos: Parâmetros murinométricos/nutricionais, ecocardiográficos e hemodinâmicos foram determinados em ratos machos com um, cinco e 12 meses de idade. A expressão de proteínas importantes na dinâmica do cálcio intracelular ena sinalização da leptina foram investigadas em homogenato de coração de rato, bem como a atividade das ATPases cardíacas. Os dados foram apresentados como média±erro-padrão e analisados pelo teste one way ANOVA (*p<0,05 vs. 1 mês e #p<0,05 vs. 5 meses). Resultados: Enquanto o índice de massa corporal aumentou (0,46±0,01 g/cm2 ; 0,75±0,01 g/cm2*; 0,78±0,01 g/cm2*), ocoeficiente de eficácia alimentar (0,431±0,013; 0,035±0,003*; 0,003±0,001*#), a velocidade máxima desenvolvida em teste de esforço (3,36±0,34 km/h; 1,38±0,04 km/h*;1,20±0,13 km/h*) e a frequência cardíaca (410,2±5,9 bpm; 375,9±7,6 bpm*;376,6±3,3 bpm*) diminuíram com a idade. Foram observadas hipertrofia do ventrículo esquerdo e disfunção diastólicaem paralelo à redução da expressão do receptor para leptina (2,1±0,4; 1,9±0,2; 0,8±0,2*#) e da atividade da bomba decálcio da família SERCA (1981±77 nmol Pi/mg de proteína/h; 2385±205 nmol Pi/mg de proteína/h; 1148±152 nmol Pi/mg de proteína/h#) no coração.Conclusões: O envelhecimento está associado a risco cardiometabólico, sendo a infrarregulação de receptores para leptina e a redução da atividade da bomba de cálcio no coração provavelmente mecanismos subjacentes à disfunção diastólica do ventrículo esquerdo e a consequente intolerância ao exercício.

Background: Aging involves physical and psychological changes that reduce the elderly’s ability to adapt themselves to society, which is the leading risk factor for cardiovascular diseases. Objective: To investigate changes in the cardiovascular system resulting from the aging process in rats. Methods: Murinometric/nutritional, echocardiographic and hemodynamic parameters were determined in 1, 5 and 12-month aged male rats. The expression of proteins that are critical to intracellular calcium dynamics and leptin signaling, as well as cardiac ATPase activity, was investigated in cardiac homogenates of rats. Data were expressed as mean ± standard error and analyzed by ANOVA one-way test (* p <0.05 vs. one month and #p <0.05 vs. 5 months). Results: Whereas the body mass index increased (0.46±0.01 g/cm2; 0.75±0.01 g/cm2 *,0.78±0.01 g/cm2*), the food efficiency ratio(0.431±0.013; 0.035±0.003*; 0.003±0.001*#), maximum speed during maximal exercise stress testing (3.36±0.34 km/h; 1.38±0.04 km/h*;1.20±0.13 km/h*) and heart rate (410.2±5.9bpm; 375.9±7.6 bpm*; 376.6±3,3 bpm*) decreased with age. Left ventricular hypertrophy and diastolic dysfunction along with reduced leptin receptor expression (2.1±0.4; 1.9±0.2; 0.8±0.2*#) and SERCA-type calcium pump activity (1981±77 nmol Pi/mg protein/h; 2385±205 nmol Pi/mg protein/h; 1148±152 nmol Pi/mg protein/h#) were observed in the hearts.Conclusions: Aging process is related to cardiometabolic risk, with cardiac leptin receptor downregulation and reduced cardiac SERCA2 calcium pump activity presumably being mechanisms underlying the left ventricular diastolic dysfunction and consequent exercise intolerance.

Modulação da diferenciação neural de células troco embrionárias por transientes de cálcio intracelulares: papéis dos receptores purinérgicos e de canais de cálcio voltagem-dependentes / Modulation of neural embryonic stem cell differentiation by intracellular Ca2+ oscillations. Roles of purinergic receptors and voltage gated Ca2+ channels

Receptores purinérgicos e canais de cálcio voltagem-dependentes estão envolvidos em diversos processos biológicos como na gastrulação, durante o desenvolvimento embrionário, e na diferenciação neural. Quando ativados, canais de cálcio voltagem-dependentes e receptores purinérgicos do tipo P2, ativados por nucleotídeos, desencadeiam transientes de cálcio intracelulares controlando diversos processos biológicos. Neste trabalho, nós estudamos a participação de canais de cálcio voltagem-dependentes e receptores do tipo P2 na geração de transientes de cálcio espontâneos e sua regulação na expressão de fatores de transcrição relacionados com a neurogênese utilizando como modelo células tronco (CTE) induzidas à diferenciação em células tronco neurais (NSC) com ácido retinóico. Descrevemos que CTE indiferenciadas podem ter a proliferação acelerada pela ativação de receptores P2X7, enquanto que a expressão e a atividade desse receptor precisam ser inibidas para o progresso da diferenciação em neuroblasto. Além disso, ao longo da diferenciação neural, por análise em tempo real dos níveis de cálcio intracelular livre identificamos 3 padrões de oscilações espontâneas de cálcio (onda, pico e unique), e mostramos que ondas e picos tiveram a frequência e amplitude aumentadas conforme o andamento da diferenciação. Células tratadas com o inibidor do receptor de inositol 1,4,5-trifosfato (IP3R), Xestospongin C, apresentaram picos mas não ondas, indicando que ondas dependem exclusivamente de cálcio oriundo do retículo endoplasmático pela ativação de IP3R. NSC de telencéfalo de embrião de camundongos transgênicos ou pré-diferenciadas de CTE tratadas com Bz-ATP, o agonista do receptor P2X7, e com 2SUTP, agonista de P2Y2 e P2Y4, aumentaram a frequência e a amplitude das oscilações espontâneas de cálcio do tipo pico. Dados, obtidos por microscopia de luminescência, da expressão em tempo real de gene repórter luciferase fusionado à Mash1 e Ngn2 revelou que a ativação dos receptores P2Y2/P2Y4 aumentou a expressão estável de Mash1 enquanto que ativação do receptor P2X7 levou ao aumento de Ngn2. Além disso, células na presença do quelante de cálcio extracelular (EGTA) ou do depletor dos estoques intracelulares de cálcio do retículo endoplasmático (thapsigargin) apresentaram redução na expressão de Mash1 e Ngn2, indicando que ambos são regulados pela sinalização de cálcio. A investigação dos canais de cálcio voltagem-dependentes demonstrou que o influxo de cálcio gerado por despolarização da membrana de NSC diferenciadas de CTE é decorrente da ativação de canais de cálcio voltagem-dependentes do tipo L. Além disso, esse influxo pode controlar o destino celular por estabilizar expressão de Mash1 e induzir a diferenciação neuronal por fosforilação e translocação do fator de transcrição CREB. Esses dados sugerem que os receptores P2X7, P2Y2, P2Y4 e canais de cálcio voltagem-dependentes do tipo L podem modular as oscilações espontâneas de cálcio durante a diferenciação neural e consequentemente alteram o padrão de expressão de Mash1 e Ngn2 favorecendo a decisão do destino celular neuronal

Purinergic receptors and voltage gated Ca2+ channels have been attributed with developmental functions including gastrulation and neural differentiation. Upon activation, nucleotide-activated P2 purinergic receptor and voltage-gated Ca2+ channel subtypes trigger intracellular calcium transients controlling cellular processes. Here, we studied the participation of voltage-gated calcium channels and P2 receptor activity in spontaneous calcium transients and consequent regulation expression of transcription factors related to retinoic acid-induced neurogenesis of mouse neural stem and embryonic stem cells (ESC). In embryonic pluripotent stem cells, proliferation is accelerated by P2X7 receptor activation, while receptor expression / activity needs to be down-regulated for the progress of neuroblast differentiation. Moreover, along neural differentiation time lapse imaging with means of a cytosolic calcium-sensitive fluorescent probe provided different patterns of spontaneous calcium transients (waves and spikes) showing that both, frequency and amplitude increased along differentiation. Cells treated with the inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor Xestospongin C showed spikes but not waves, indicating that waves exclusively depended on calcium release from endoplasmic reticulum by IP3R activation. Cells treated with the P2X7 receptor subtype agonist Bz-ATP and the P2Y2 and P2Y4 receptor 2-S-UTP increased frequency and amplitudes of calcium transients, mainly spikes, in embryonic telencephalon neural stem cells (NSC) and NSC pre-differentiated from ESC. Data obtained by luminescence time lapse imaging of stable transfected cells with Mash1 or Ngn2 promoter-protein fusion to luciferase reporter construct revealed increased Mash1 expression due to activation of P2Y2/P2Y4 receptor subtypes, while increased expression of Ngn2 was observed following P2X7 receptor activation. In addition, cells imaged in presence of the extracellular calcium chelator EGTA or following endoplasmic reticulum calcium store depletion by thapsigargin showed a decrease in Mash1 and Ngn2 expression, indicating that both are regulated by calcium signaling. Investigation of the roles of voltage gated Ca2+ channels in neural differentiation showed that Ca2+ influx in NSC pre-differentiated from ESC is due to membrane depolarization and L-type voltage gated Ca2+ channel activation, thereby controlling cell fate decision, by stabilizing the expression of MASH1 and inducing differentiation, by phosphorylation of the transcription factor CREB. Altogether these data suggest that P2X7, P2Y2, P2Y4 receptors and L-type voltage gated Ca2+ channels can modulate spontaneous calcium oscillations during neural differentiation and consequently change the Mash1 and Ngn2 expression patterns, thus favoring the cell fate decision to the neuronal phenotype

Effect of exercise training on Ca2+ release units of left ventricular myocytes of spontaneously hypertensive rats

In cardiomyocytes, calcium (Ca2+) release units comprise clusters of intracellular Ca2+ release channels located on the sarcoplasmic reticulum, and hypertension is well established as a cause of defects in calcium release unit function. Our objective was to determine whether endurance exercise training could attenuate the deleterious effects of hypertension on calcium release unit components and Ca2+ sparks in left ventricular myocytes of spontaneously hypertensive rats. Male Wistar and spontaneously hypertensive rats (4 months of age) were divided into 4 groups: normotensive (NC) and hypertensive control (HC), and normotensive (NT) and hypertensive trained (HT) animals (7 rats per group). NC and HC rats were submitted to a low-intensity treadmill running protocol (5 days/week, 1 h/day, 0% grade, and 50-60% of maximal running speed) for 8 weeks. Gene expression of the ryanodine receptor type 2 (RyR2) and FK506 binding protein (FKBP12.6) increased (270%) and decreased (88%), respectively, in HC compared to NC rats. Endurance exercise training reversed these changes by reducing RyR2 (230%) and normalizing FKBP12.6 gene expression (112%). Hypertension also increased the frequency of Ca2+ sparks (HC=7.61±0.26 vs NC=4.79±0.19 per 100 µm/s) and decreased its amplitude (HC=0.260±0.08 vs NC=0.324±0.10 ΔF/F0), full width at half-maximum amplitude (HC=1.05±0.08 vs NC=1.26±0.01 µm), total duration (HC=11.51±0.12 vs NC=14.97±0.24 ms), time to peak (HC=4.84±0.06 vs NC=6.31±0.14 ms), and time constant of decay (HC=8.68±0.12 vs NC=10.21±0.22 ms). These changes were partially reversed in HT rats (frequency of Ca2+ sparks=6.26±0.19 µm/s, amplitude=0.282±0.10 ΔF/F0, full width at half-maximum amplitude=1.14±0.01 µm, total duration=13.34±0.17 ms, time to peak=5.43±0.08 ms, and time constant of decay=9.43±0.15 ms). Endurance exercise training attenuated the deleterious effects of hypertension on calcium release units of left ventricular myocytes.

Preputial gland activates olfactory receptor neurons in rat: Calcium imaging study using laser scanning confocal microscopy.

The rodent preputial gland is one of the major sources of odours and is reported to be involved in several behavioural activities. However, how the preputial gland initiates the olfactory response to manifest the effects is not known. Olfactory receptor neurons (ORNs) present in the olfactory epithelium are involved in the perception of odorant/pheromonal compounds. In the present study, the response of rat ORNs to preputial gland extract was evaluated by calcium imaging analysis. We found that some rat ORNs responded to the preputial gland extract by exhibiting an intracellular calcium response. By contrast, the ORNs did not respond at all to the foot pad extract (control). The results indicated that the substances contained in the preputial gland might interact with a type of receptor expressed in the female rat ORNs, suggested to manifest the behavioural responses, such as social and sexual interactions. This study provided the first evidence of activation of ORNs by the preputial gland extract.

El Ca2+ y los esfingolípidos como moduladores de la apoptosis y el cáncer / Ca2+ and sphingolipids as modulators for apoptosis and cancer

El Ca2+ es un segundo mensajero que regula funciones directamente relacionadas con el cáncer como la proliferación, diferenciación y la apoptosis. La concentración intracelular de Ca2+ ([Ca2+]i) está altamente regulada por diversos mecanismos entre los que destacan canales iónicos, la Ca2+-ATPasa del retículo endoplasmático (SERCA) y de la membrana plasmática (PMCA), y el transporte de Ca2+ mitocondrial. En el cáncer, la célula tumoral prolifera sin control tras su incapacidad de reconocer señales apoptóticas. La apoptosis es mediada a través de cambios en la actividad de ciertas proteínas como las caspasas y miembros de la familia Bcl-2. Adicionalmente, el “estrés del retículo”, promovido por la acumulación y agregación de proteínas mal plegadas en el interior del retículo endoplasmático (RE), puede desencadenar la apoptosis. El “estrés del retículo” es inducido por una variedad de agentes, entre los que destaca la tapsigargina, inhibidor específico de la SERCA, la cual promueve un notable aumento en la [Ca2+]i, induciendo además apoptosis. En consecuencia, actualmente se están ensayando exitosamente derivados de la tapsigargina como agentes antineoplásicos. El Ca2+ es transferido a la mitocondria desencadenando señales apoptóticas. Por otra parte, los esfingolípidos, como la ceramida y la esfingosina, y sus derivados fosforilados, la ceramida-1-fosfato y la esfingosina-1-fosfato, los cuales regulan la [Ca2+]i, también están estrechamente vinculados con la señalización intracelular en procesos relacionados con el cáncer. Esta revisión discute nuevas evidencias sobre el efecto de estos esfingolípidos en la homeostasis de Ca+2 intracelular y su conexión con la apoptosis y el cáncer.

Ca2+ is a second messenger which regulates many functions directly related with cancer such as proliferation, differentiation and apoptosis. The intracellular Ca2+ concentration ([Ca2+]i) is finely regulated by several mechanisms, among them ionic channels, the endoplasmic reticulum Ca2+-ATPase (SERCA), the plasma membrane calcium pump (PMCA) and the mitochondrial Ca2+ transport. In cancer, the tumour cell proliferates without control since the capacity to recognize apoptotic signals has been lost. The apoptosis is regulated by changes in several proteins, as caspases and the Bcl-2 family members, among others. Additionally, the “reticulum stress”, promoted by the accumulation and aggregation of unfolded proteins in the interior of the endoplasmic reticulum (ER), ussually leads to apoptosis. The “reticulum stress” can be induced by several agents, remarkably with thapsigargin, a selective inhibitor of the SERCA, which in turn induces a large increment in [Ca2+]I, leading to apoptosis. As a consequence, currently, derivatives of thapsigargin are successfully been assayed as anti-neoplastic agents. Ca2+ is then transferred to the mitochondria, where it is known to constitute a main apoptotic signal. On the other hand, several sphingolipids, such as ceramide and sphingosine, and their phosphorylated derivatives ceramide-1-phosphate and sphingosine-1-phosphate, directly involved in the [Ca2+]I regulation, are also recognized as signal messengers related with cancer processes. In this review we discuss new evidences on the effect of several sphingolipids in the intracellular Ca2+ homeostasis and its relationship with apoptosis and cancer.

STIM1/Orai1-mediated store-operated Ca2+ entry: the tip of the iceberg

Highly efficient mechanisms regulate intracellular calcium (Ca2+) levels. The recent discovery of new components linking intracellular Ca2+ stores to plasma membrane Ca2+ entry channels has brought new insight into the understanding of Ca2+ homeostasis. Stromal interaction molecule 1 (STIM1) was identified as a Ca2+ sensor essential for Ca2+ store depletion-triggered Ca2+ influx. Orai1 was recognized as being an essential component for the Ca2+ release-activated Ca2+ (CRAC) channel. Together, these proteins participate in store-operated Ca2+ channel function. Defective regulation of intracellular Ca2+ is a hallmark of several diseases. In this review, we focus on Ca2+ regulation by the STIM1/Orai1 pathway and review evidence that implicates STIM1/Orai1 in several pathological conditions including cardiovascular and pulmonary diseases, among others.

Lack of evidence for regulation of cardiac P-type ATPases and MAP kinases in transgenic mice with cardiac-specific overexpression of constitutively active á1B-adrenoceptors

The regulatory function of á1B-adrenoceptors in mammalian heart homeostasis is controversial. The objective of the present study was to characterize the expression/activity of key proteins implicated in cardiac calcium handling (Na+/K+-ATPase and Ca2+-ATPases) and growth (ERK1/2, JNK1/2 and p38) in mice with cardiac-selective overexpression of constitutively active mutant á1B-adrenoceptor (CAMá1B-AR), which present a mild cardiac hypertrophy phenotype. Immunoblot assays showed that myocardial plasma membrane Ca2+-ATPase (PMCA) expression was increased by 30 percent in CAMá1B-AR mice (N = 6, P < 0.05), although there was no change in sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) expression. Moreover, total Ca2+-ATPase activity was not modified, but a significant increase in the activity of the thapsigargin-resistant (PMCA) to thapsigargin-sensitive (SERCA) ratio was detected. Neither Na+/K+-ATPase activity nor the expression of á1 and á2 subunit isoforms was changed in CAMá1B-AR mouse hearts. Moreover, immunoblot assays did not provide evidence for an enhanced activation of the three mitogen-activated protein kinases studied in this stage of hypertrophy. Therefore, these findings indicate that chronic cardiac á1B-AR activation in vivo led to mild hypertrophy devoid of significant signs of adaptive modifications concerning primary intracellular calcium control and growth-related proteins, suggesting a minor pathophysiological role of this adrenergic receptor in mouse heart at this stage of development.

Calcium and cell death signaling in neurodegeneration and aging

Transient increase in cytosolic (Cac2+) and mitochondrial Ca2+ (Ca m2+) are essential elements in the control of many physiological processes. However, sustained increases in Ca c2+ and Ca m2+ may contribute to oxidative stress and cell death. Several events are related to the increase in Ca m2+, including regulation and activation of a number of Ca2+ dependent enzymes, such as phospholipases, proteases and nucleases. Mitochondria and endoplasmic reticulum (ER) play pivotal roles in the maintenance of intracellular Ca2+ homeostasis and regulation of cell death. Several lines of evidence have shown that, in the presence of some apoptotic stimuli, the activation of mitochondrial processes maylead to the release of cytochrome c followed by the activation of caspases, nuclear fragmentation and apoptotic cell death. The aim of this review was to show how changes in calcium signaling can be related to the apoptotic cell death induction. Calcium homeostasis was also shown to be an important mechanism involved in neurodegenerative and aging processes.

Aumentos transientes no cálcio citosólico (Ca c2+) e mitocondrial (Ca m2+) são elementos essenciais no controle de muitos processos fisiológicos. No entanto, aumentos sustentados do Ca c2+ e do Ca m2+ podem contribuir para o estresse oxidativo ea morte celular. Muitos eventos estão relacionados ao aumentono Ca c2+, incluindo a regulação e ativação de várias enzimas dependentes de Ca2+ como as fosfolipases, proteases e nucleases. A mitocôndria e o retículo endoplasmático têm um papel central na manutenção da homeostase intracellular de Ca c2+ e na regulação da morte celular. Várias evidências mostraram que, na presença de certos estímulos apoptóticos, a ativação dos processos mitocondriais pode promover a liberação de citocromo c, seguida da ativação de caspases, fragmentação nuclear e morte celular por apoptose. O objetivo desta revisão é mostrar como aumentos na sinalização de Ca2+ podem estar relacionados aos eventos de indução da morte celular apoptótica. Além disso, evidenciar como a homeostase de Ca2+ pode ser importante e está envolvida nos mecanismos presentes nos processos de neurodegeneração e envelhecimento.

Nuclear calcium signaling: a cell within a cell

Calcium (Ca2+) is a versatile second messenger that regulates a wide range of cellular functions. Although it is not established how a single second messenger coordinates diverse effects within a cell, there is increasing evidence that the spatial patterns of Ca2+ signals may determine their specificity. Ca2+ signaling patterns can vary in different regions of the cell and Ca2+ signals in nuclear and cytoplasmic compartments have been reported to occur independently. No general paradigm has been established yet to explain whether, how, or when Ca2+ signals are initiated within the nucleus or their function. Here we highlight that receptor tyrosine kinases rapidly translocate to the nucleus. Ca2+ signals that are induced by growth factors result from phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate formation within the nucleus rather than within the cytoplasm. This novel signaling mechanism may be responsible for growth factor effects on cell proliferation.

Synaptic vesicle pool size, release probability and synaptic depression are sensitive to Ca2+ buffering capacity in the developing rat calyx of Held

The calyx of Held, a specialized synaptic terminal in the medial nucleus of the trapezoid body, undergoes a series of changes during postnatal development that prepares this synapse for reliable high frequency firing. These changes reduce short-term synaptic depression during tetanic stimulation and thereby prevent action potential failures during a stimulus train. We measured presynaptic membrane capacitance changes in calyces from young postnatal day 5-7 (p5-7) or older (p10-12) rat pups to examine the effect of calcium buffer capacity on vesicle pool size and the efficiency of exocytosis. Vesicle pool size was sensitive to the choice and concentration of exogenous Ca2+ buffer, and this sensitivity was much stronger in younger animals. Pool size and exocytosis efficiency in p5-7 calyces were depressed by 0.2 mM EGTA to a greater extent than with 0.05 mM BAPTA, even though BAPTA is a 100-fold faster Ca2+ buffer. However, this was not the case for p10-12 calyces. With 5 mM EGTA, exocytosis efficiency was reduced to a much larger extent in young calyces compared to older calyces. Depression of exocytosis using pairs of 10-ms depolarizations was reduced by 0.2 mM EGTA compared to 0.05 mM BAPTA to a similar extent in both age groups. These results indicate a developmentally regulated heterogeneity in the sensitivity of different vesicle pools to Ca2+ buffer capacity. We propose that, during development, a population of vesicles that are tightly coupled to Ca2+ channels expands at the expense of vesicles more distant from Ca2+ channels.

Ca2+ signaling in pancreatic acinar cells: physiology and pathophysiology

The pancreatic acinar cell is a classical model for studies of secretion and signal transduction mechanisms. Because of the extensive endoplasmic reticulum and the large granular compartment, it has been possible - by direct measurements - to obtain considerable insights into intracellular Ca2+ handling under both normal and pathological conditions. Recent studies have also revealed important characteristics of stimulus-secretion coupling mechanisms in isolated human pancreatic acinar cells. The acinar cells are potentially dangerous because of the high intra-granular concentration of proteases, which become inappropriately activated in the human disease acute pancreatitis. This disease is due to toxic Ca2+ signals generated by excessive liberation of Ca2+ from both the endoplasmic reticulum and the secretory granules.

Aspectos fisiológicos do cálcio e sua disfunção em patologias neurodegenerativas / Physiologic aspects of calcium and your dysfunction in neurodegenerative pathologies

O íon cálcio funciona como um segundo mensageiro que regula um amplo espectro de processos celulares. A diminuição ou perda do controle dos mecanismos que regulam a concentração intracelular desse íon está associada, respectivamente, ao envelhecimento dos neurônios e a doenças neurodegenerativas. A gênese dessas modificações é desconhecida. Entretanto, estudos recentes apontam para uma provável correlação entre expressão gênica alterada, estresse do retículo endoplasmático e os processos patológicos associados à disfunção na concentração intracelular do cálcio. O esclarecimento dessas questões poderá trazer novos alvos terapêuticos capazes de frear ou reverter tais alterações, combatendo, dessa forma, tanto o envelhecimento neuronal quanto as doenças neurodegenerativas.

Calcium is a second messenger that regulates a lot of cellular functions. The following mechanisms regulate the intracellular concentrations of the ion: influx, release, extrusion and storage. Decrease or loss in control of these mechanisms is related to aging of neurons and neurodegenerative diseases, respectively. The genesis of these alterations is unknown. However, recent studies point to a correlation between calcium dysfunction and altered gene expression. There is also a correlation between endoplasmic reticulum stress and pathological processes. Further investigations may reveal new therapeutical targets that can block or revert these alterations.

Modularized study of human calcium signalling pathway.

Signalling pathways are complex biochemical networks responsible for regulation of numerous cellular functions. These networks function by serial and successive interactions among a large number of vital biomolecules and chemical compounds. For deciphering and analysing the underlying mechanism of such networks,a modularized study is quite helpful. Here we propose an algorithm for modularization of calcium signalling pathway of H. sapiens .The idea that "a node whose function is dependent on maximum number of other nodes tends to be the center of a sub network" is used to divide a large signalling network into smaller sub networks. Inclusion of node(s) into sub networks(s) is dependent on the outdegree of the node(s). Here outdegree of a node refers to the number of relations of the considered node lying outside the constructed sub network. Node(s) having more than c relations lying outside the expanding sub network have to be excluded from it. Here c is a specified variable based on user preference, which is finally fixed during adjustments of created sub networks, so that certain biological significance can be conferred on them.

Avaliação morfológica e dos mecanismos de mobilização de Ca2+ pela glicose e acetilcolina em células pancreáticas humanas / Morphologic evaluation and Ca2+ mobilization by glicose and acetylcholine in human pancreatic cells

OBJETIVOS: Avaliar a morfologia das organelas e do citoesqueleto em células pancreáticas humanas cultivadas e a mobilização de Ca2+ em resposta à glicose e ACh por medidas fluorimétricas. MATERIAL E MÉTODOS: As células foram semeadas em lamínulas, fixadas e marcadas com uma combinação de fluoróforos: o núcleo foi corado com DAPI e as mitocôndrias, com Mytotracker Red. Foram utilizados faloidina e anticorpos secundários conjugados com Alexa Fluor verde e vermelho fluorescentes (488 e 594) para identificar proteína actina F e receptor muscarínico tipo M3, respectivamente. Para estudar a mobilização de Ca2+, as células foram incubadas com fura-2/AM. RESULTADOS: As células pancreáticas humanas apresentaram morfologia preservada com grande quantidade de mitocôndrias. Na região de maior densidade celular, evidenciou-se as pseudo-ilhotas e os receptores muscarínicos M3. Por meio da elevação da [Ca2+]c, devido à ação da glicose e ACh, mostrou-se preservação da capacidade responsiva a esses estímulos e foi dependente de concentração desses agonistas. A glicose promoveu uma resposta sustentada e a ACh induziu uma resposta bifásica. CONCLUSÃO: As células pancreáticas humanas cultivadas conservaram sua morfologia. A mobilização de Ca2+ em resposta à glicose e a ACh confirma a sua funcionalidade. Os receptores muscarínicos M3 estão presentes nessas células.

AIMS: The proposal of this study was to analyze morphology of the organelles and cytoskeleton in human pancreatic cells cultured and the mobilization of the cytosolic calcium ([Ca2+]c) in response to glucose and ACh by fluorimetry method. MATERIAL AND METHODS: The cells were plated on glass coverslips, fixed and stained with a combination of fluorophores: the nuclei were stained with DAPI and mitochondria with Mytotracker Red. It was used phalloidin and the secondary antibodies Alexa Fluor conjugated green and red-fluorescent (488 and 594) to identify the protein cell actin F and type M3 muscarinic receptor respectively. The cells also were loaded with fura-2/AM to study Ca2+ mobilization. RESULTS: The human pancreatic cells show characteristics morphologically preserved with great amount of mitochondria. In region major cell density was evidenced pseudo-islets and type M3 muscarinic receptors. Through increase of [Ca2+]c due to action of glucose and ACh were shown that the cellsÆ capacity to respond to these stimuli were conserved. The elevation of the [Ca2+]c depended on concentration by glucose-induced promoting sustained phase and ACh-induced a biphasic response. CONCLUSION: The morphologic characteristics of human pancreatic cells cultured were preserved. The Ca2+ mobilization in response to glucose and ACh confirmed its functionality. The expression of the M3 muscarinic receptors in human pancreatic cell cultured was demonstrated.

Molecular basis of mammalian cell invasion by Trypanosoma cruzi

O estabelecimento da infecção por Trypanosoma cruzi, o agente da doença de Chagas, depende de uma série de eventos envolvendo interações de diversas moléculas do parasita com componentes do hospedeiro. Focalizamos aqui os mecanismos de invasão celular por tripomastigotas metacíclicos (TM) e por tripomastigotas de cultura de tecido (TCT). Durante a internalização de TM ou TCT, vias de transdução de sinal são ativadas tanto no parasita como na célula alvo, acarretando a mobilização de Ca2+. Para adesão, TM utiliza as glicoproteínas de superfície como a gp82 e gp35/50, que são moléculas indutoras de sinal de Ca2+. Em isolados de T. cruzi que entram na célula hospedeira de maneira dependente de gp82, a proteína tirosina quinase assim como a fosfolipase C do parasita são ativadas, e Ca2+ é liberado de reservatórios sensíveis a IP3, enquanto em isolados de T. cruzi que se ligam às células alvo através de gp35/50, a via de sinalização envolvendo adenilil ciclase parece ser estimulada, com liberação de Ca2+ de acidocalciossomos. Além disso, dependendo do isolado de T. cruzi, sinais inibitórios mediados por gp90 específica de TM podem ser desencadeados tanto na célula hospedeira como no parasita. O repertório de moléculas de TCT implicadas na invasão celular inclui glicoproteínas de superfície da família gp85, com membros contendo sitos de ligação à laminina e citoqueratina 18, enzimas como a cruzipaína, trans-sialidase, e uma oligopeptidase B que gera um agonista de Ca2+ a partir de uma molécula precursora. .

Pituitary cell type-specific electrical activity, calcium signaling and secretion

All secretory anterior pituitary cells exhibit spontaneous and extracellular calcium-dependent electrical activity, but differ with respect to the patterns of firing and associated calcium signaling and hormone secretion. Thus, somatotrophs and lactotrophs fire plateau-bursting action potentials spontaneously and without coupling to calcium release from intracellular stores, which generate calcium signals of sufficient amplitude to keep steady hormone release. In these cells, both spontaneous electrical activity and basal hormone secretion can be further amplified by activation of Gq/11 and Gs-coupled receptors and inhibited by Gi/o/z-coupled receptors. In contrast, gonadotrophs fire single, high-amplitude spikes with limited ability to promote calcium influx and exocytosis, whereas activated Gq/11-coupled receptors in these cells transform single-action potential spiking into the plateau-bursting type of electrical activity and trigger periodic high-amplitude calcium signals and exocytosis of prestored secretory vesicles. Here, we review biochemical and biophysical aspects of spontaneous and receptor-controlled electrical activity, calcium signaling, and hormone secretion in pituitary cells.

Effect of the zinc chelator N, N, N',N'- tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) on hippocampal mossy fiber calcium signals and on synaptic transmission

An important pool of chelatable zinc is present in the synaptic vesicles of mossy fiber terminals from hippocampal CA3 area, being zinc released following single or repetitive electrical stimulation. Previous studies have suggested different synaptic roles for released mossy fiber zinc, including the inhibition of presynaptic calcium and of postsynaptic N-methyl-D-aspartate (NMDA) and gamma amino-butiric acid (GABA A) receptors. The effect of endogenously released zinc on mossy fiber long-term potentiation (LTP) induction also is not yet established. We have investigated the effect of the permeant zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) on mossy fiber calcium and on synaptic transmission, before and during the application of LTP-inducing stimulation. We have found, using the calcium indicator Fura-2, that single and tetanically-evoked mossy fiber calcium signals are both enhanced in the presence of 20 ìM TPEN, while the single field potentials are unaffected. As expected, no effect was observed on the single calcium signals or field potentials obtained at the CA3-CA1 synapses, from the CA1 area, which has a lower concentration of vesicular zinc. These results support the idea that at the hippocampal mossy fiber synapses, released zinc inhibits presynaptic calcium mechanisms. A higher concentration of TPEN (100 ìM) significantly reduced mossy fiber synaptic transmission but did not prevent the induction of mossy fiber LTP, suggesting that zinc is not required for the formation of this form of LTP.

Single-channel recording of inositol trisphosphate receptor in the isolated nucleus of a muscle cell line

Nuclear calcium appears to have an important role in the regulation of gene expression in many cells, but the mechanisms involved in controlling nuclear Ca2+ signaling are controversial and still poorly understood. We have described the presence of inositol 1,4,5 trisphosphate (IP3) receptors in the nuclei of skeletal muscle cells. Now, we have characterized the properties of the IP3 receptors channels present in the nuclei of the 1B5 cell line, which do not express any isoforms of the ryanodine receptor. Immunocytochemistry of isolated nuclei confirmed the presence of IP3R in the nuclear envelope and fluorescence measurements in nuclei suspensions allowed us to document ATP-dependent calcium loading by the nucleus and its release upon IP3 addition. Patch clamp of nuclear membranes was performed, and single-channel activity recorded was dependent on the presence of IP3 in the pipette; single-channel conductance was in the range reported in the literature for these channels, and the open probability was shown to be dependent on IP3 concentration.