An acid-sensing ion channel that detects ischemic pain

Ischemic pain occurs when there is insufficient blood flow for the metabolic needs of an organ. The pain of a heart attack is the prototypical example. Multiple compounds released from ischemic muscle likely contribute to this pain by acting on sensory neurons that innervate muscle. One such compound is lactic acid. Here, we show that ASIC3 (acid-sensing ion channel #3) has the appropriate expression pattern and physical properties to be the detector of this lactic acid. In rats, it is expressed only in sensory neurons and then only on a minority (~40%) of these. Nevertheless, it is expressed at extremely high levels on virtually all dorsal root ganglion sensory neurons that innervate the heart. It is extraordinarily sensitive to protons (Hill slope 4, half-activating pH 6.7), allowing it to readily respond to the small changes in extracellular pH (from 7.4 to 7.0) that occur during muscle ischemia. Moreover, both extracellular lactate and extracellular ATP increase the sensitivity of ASIC3 to protons. This final property makes ASIC3 a "coincidence detector" of three molecules that appear during ischemia, thereby allowing it to better detect acidosis caused by ischemia than other forms of systemic acidosis such as hypercapnia.

Patch-clamp detection of macromolecular translocation along nuclear pores

The present paper reviews the application of patch-clamp principles to the detection and measurement of macromolecular translocation along the nuclear pores. We demonstrate that the tight-seal 'gigaseal' between the pipette tip and the nuclear membrane is possible in the presence of fully operational nuclear pores. We show that the ability to form a gigaseal in nucleus-attached configurations does not mean that only the activity of channels from the outer membrane of the nuclear envelope can be detected. Instead, we show that, in the presence of fully operational nuclear pores, it is likely that the large-conductance ion channel activity recorded derives from the nuclear pores. We conclude the technical section with the suggestion that the best way to demonstrate that the nuclear pores are responsible for ion channel activity is by showing with fluorescence microscopy the nuclear translocation of ions and small molecules and the exclusion of the same from the cisterna enclosed by the two membranes of the envelope. Since transcription factors and mRNAs, two major groups of nuclear macromolecules, use nuclear pores to enter and exit the nucleus and play essential roles in the control of gene activity and expression, this review should be useful to cell and molecular biologists interested in understanding how patch-clamp can be used to quantitate the translocation of such macromolecules into and out of the nucleus

ClC-5 chloride channel and kidney stones: what is the link?

Nephrolithiasis is one of the most common diseases in the Western world. The disease manifests itself with intensive pain, sporadic infections, and, sometimes, renal failure. The symptoms are due to the appearance of urinary stones (calculi) which are formed mainly by calcium salts. These calcium salts precipitate in the renal papillae and/or within the collecting ducts. Inherited forms of nephrolithiasis related to chromosome X (X-linked hypercalciuric nephrolithiasis or XLN) have been recently described. Hypercalciuria, nephrocalcinosis, and male predominance are the major characteristics of these diseases. The gene responsible for the XLN forms of kidney stones was cloned and characterized as a chloride channel called ClC-5. The ClC-5 chloride channel belongs to a superfamily of voltage-gated chloride channels, whose physiological roles are not completely understood. The objective of the present review is to identify recent advances in the molecular pathology of nephrolithiasis, with emphasis on XLN. We also try to establish a link between a chloride channel like ClC-5, hypercalciuria, failure in urine acidification and protein endocytosis, which could explain the symptoms exhibited by XLN patients.

Nitric oxide modulation of the hypothalamo-neurohypophyseal system

Nitric oxide (NO), a free radical gas produced endogenously from the amino acid L-arginine by NO synthase (NOS), has important functions in modulating vasopressin and oxytocin secretion from the hypothalamo-neurohypophyseal system. NO production is stimulated during increased functional activity of magnocellular neurons, in parallel with plastic changes of the supraoptic nucleus (SON) and paraventricular nucleus. Electrophysiological data recorded from the SON of hypothalamic slices indicate that NO inhibits firing of phasic and non-phasic neurons, while L-NAME, an NOS inhibitor, increases their activity. Results from measurement of neurohypophyseal hormones are more variable. Overall, however, it appears that NO, tonically produced in the forebrain, inhibits vasopressin and oxytocin secretion during normovolemic, isosmotic conditions. During osmotic stimulation, dehydration, hypovolemia and hemorrhage, as well as high plasma levels of angiotensin II, NO inhibition of vasopressin neurons is removed, while that of oxytocin neurons is enhanced. This produces a preferential release of vasopressin over oxytocin important for correction of fluid imbalance. During late pregnancy and throughout lactation, fluid homeostasis is altered and expression of NOS in the SON is down- and up-regulated, respectively, in parallel with plastic changes of the magnocellular system. NO inhibition of magnocellular neurons involves GABA and prostaglandin synthesis and the signal-transduction mechanism is independent of the cGMP-pathway. Plasma hormone levels are unaffected by icv 1H-[1, 2, 4]oxadiazolo-[4,3-a]quinoxalin-1-one (a soluble guanylyl cyclase inhibitor) or 8-Br-cGMP administered to conscious rats. Moreover, cGMP does not increase in homogenates of the neural lobe and in microdialysates of the SON when NO synthesis is enhanced during osmotic stimulation. Among alternative signal-transduction pathways, nitrosylation of target proteins affecting activity of ion channels is considered.

Reactive oxygen species and angiotensin II signaling in vascular cells: implications in cardiovascular disease

Diseases such as hypertension, atherosclerosis, hyperlipidemia, and diabetes are associated with vascular functional and structural changes including endothelial dysfunction, altered contractility and vascular remodeling. Cellular events underlying these processes involve changes in vascular smooth muscle cell (VSMC) growth, apoptosis/anoikis, cell migration, inflammation, and fibrosis. Many factors influence cellular changes, of which angiotensin II (Ang II) appears to be amongst the most important. The physiological and pathophysiological actions of Ang II are mediated primarily via the Ang II type 1 receptor. Growing evidence indicates that Ang II induces its pleiotropic vascular effects through NADPH-driven generation of reactive oxygen species (ROS). ROS function as important intracellular and intercellular second messengers to modulate many downstream signaling molecules, such as protein tyrosine phosphatases, protein tyrosine kinases, transcription factors, mitogen-activated protein kinases, and ion channels. Induction of these signaling cascades leads to VSMC growth and migration, regulation of endothelial function, expression of pro-inflammatory mediators, and modification of extracellular matrix. In addition, ROS increase intracellular free Ca2+ concentration ([Ca2+]i), a major determinant of vascular reactivity. ROS influence signaling molecules by altering the intracellular redox state and by oxidative modification of proteins. In physiological conditions, these events play an important role in maintaining vascular function and integrity. Under pathological conditions ROS contribute to vascular dysfunction and remodeling through oxidative damage. The present review focuses on the biology of ROS in Ang II signaling in vascular cells and discusses how oxidative stress contributes to vascular damage in cardiovascular disease.

Modulation of store-operated Ca2+ entry by cyclic-ADP-ribose

Store-operated Ca2+ entry plays an important role in Ca2+ homeostasis in cells but the mechanisms of control of these channels are not completely understood. We describe an investigation of the role of the CD38-cyclic-ADP-ribose (cADPR)-ryanodine-channel (RyR) signaling pathway in store-operated Ca2+ entry in human smooth muscle. We observed that human myometrial cells have a functional store-operated Ca2+ entry mechanism. Furthermore, we observed the presence of transient receptor potential 1, 3, 4, 5, and 6 ion channels in human myometrial cells. Store-operated Ca2+ transient was inhibited by at least 50-70% by several inhibitors of the RyR, including ryanodine (10 µM), dantrolene (10 µM), and ruthenium red (10 µM). Furthermore, the cell permeable inhibitor of the cADPR-system, 8-Br-cADPR (100 µM), is a potent inhibitor of the store-operated entry, decreasing the store operated entry by 80%. Pre-incubation of cells with 100 µM cADPR and the hydrolysis-resistant cADPR analog 3-deaza-cADPR (50 µM), but not with ADP-ribose (ADPR) leads to a 1.6-fold increase in the store-operated Ca2+ transient. In addition, we observed that nicotinamide (1-10 mM), an inhibitor of cADPR synthesis, also leads to inhibition of the store-operated Ca2+ transient by 50-80%. Finally, we observed that the transient receptor potential channels, RyR, and CD38 can be co-immunoprecipitated, indicating that they interact in vivo. Our observations clearly implicate the CD38-cADPR-ryanodine signaling pathway in the regulation of store-operated Ca2+ entry in human smooth muscle cells.

Involvement of the TRPV1 channel in the modulation of spontaneous locomotor activity, physical performance and physical exercise-induced physiological responses

Physical exercise triggers coordinated physiological responses to meet the augmented metabolic demand of contracting muscles. To provide adequate responses, the brain must receive sensory information about the physiological status of peripheral tissues and organs, such as changes in osmolality, temperature and pH. Most of the receptors involved in these afferent pathways express ion channels, including transient receptor potential (TRP) channels, which are usually activated by more than one type of stimulus and are therefore considered polymodal receptors. Among these TRP channels, the TRPV1 channel (transient receptor potential vanilloid type 1 or capsaicin receptor) has well-documented functions in the modulation of pain sensation and thermoregulatory responses. However, the TRPV1 channel is also expressed in non-neural tissues, suggesting that this channel may perform a broad range of functions. In this review, we first present a brief overview of the available tools for studying the physiological roles of the TRPV1 channel. Then, we present the relationship between the TRPV1 channel and spontaneous locomotor activity, physical performance, and modulation of several physiological responses, including water and electrolyte balance, muscle hypertrophy, and metabolic, cardiovascular, gastrointestinal, and inflammatory responses. Altogether, the data presented herein indicate that the TPRV1 channel modulates many physiological functions other than nociception and thermoregulation. In addition, these data open new possibilities for investigating the role of this channel in the acute effects induced by a single bout of physical exercise and in the chronic effects induced by physical training.

Skin secretion of Siphonops paulensis (Gymnophiona, Amphibia) forms voltage-dependent ionic channels in lipid membranes

The effect of the skin secretion of the amphibian Siphonops paulensis was investigated by monitoring the changes in conductance of an artificial planar lipid bilayer. Skin secretion was obtained by exposure of the animals to ether-saturated air, and then rinsing the animals with distilled water. Artificial lipid bilayers were obtained by spreading a solution of azolectin over an aperture of a Delrin cup inserted into a cut-away polyvinyl chloride block. In 9 of 12 experiments, the addition of the skin secretion to lipid bilayers displayed voltage-dependent channels with average unitary conductance of 258 ± 41.67 pS, rather than nonspecific changes in bilayer conductance. These channels were not sensitive to 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid or tetraethylammonium ion, but the experimental protocol used does not permit us to specify their characteristics.

Monetary channels of social inclusion: a case study of basic income and the Caixa Econômica Federal in Brazil

This article reports evidence of new monetary channels for social inclusion involving basic income policies and the Caixa Econômica Federal, a Brazilian government savings bank. Since the Plano Real (Brazilian currency) and the liberalization of banking in the 1990s, the realization of competitive advantages by the Caixa as social policy agent and the importance of citizenship cards differ from existing theories of bank change, financial inclusion and monetary policy. Multi-method research reveals the importance of 1) political theories of basic income, 2) conceptions of citizenship and social justice, and 3) a back to the future modernization of government banking. This provides alternatives to contemporary market-based banking theory, neo-liberal policies, private and non-governmental microfinance strategies, and theories in political economy about fiscal constraints to social policies. New monetary channels of change also suggest that zero sum theories about politics, monetary authority and social inclusion are amiss.

On line pre-concentration for simultaneous determination of low molecular weight organic acids and inorganic anions in Amazonian river water samples employing ion chromatography with conductivity detection

An ion chromatography procedure, employing an IonPac AC15 concentrator column was used to investigate on line preconcentration for the simultaneous determination of inorganic anions and organic acids in river water. Twelve organic acids and nine inorganic anions were separated without any interference from other compounds and carry-over problems between samples. The injection loop was replaced by a Dionex AC15 concentrator column. The proposed procedure employed an auto-sampler that injected 1.5 ml of sample into a KOH mobile phase, generated by an Eluent Generator, at 1.5 mL min-1, which carried the sample to the chromatographic columns (one guard column, model AG-15, and one analytical column, model AS15, with 250 x 4mm i.d.). The gradient elution concentrations consisted of a 10.0 mmol l-1 KOH solution from 0 to 6.5 min, gradually increased to 45.0 mmol l-1 KOH at 21 min., and immediatelly returned and maintained at the initial concentrations until 24 min. of total run. The compounds were eluted and transported to an electro-conductivity detection cell that was attached to an electrochemical detector. The advantage of using concentrator column was the capability of performing routine simultaneous determinations for ions from 0.01 to 1.0 mg l-1 organic acids (acetate, propionic acid, formic acid, butyric acid, glycolic acid, pyruvate, tartaric acid, phthalic acid, methanesulfonic acid, valeric acid, maleic acid, oxalic acid, chlorate and citric acid) and 0.01 to 5.0 mg l-1 inorganic anions (fluoride, chloride, nitrite, nitrate, bromide, sulfate and phosphate), without extensive sample pretreatment and with an analysis time of only 24 minutes.

Diferenças relacionadas ao sexo nos volumes ventriculares e na fração de ejeção do ventrículo esquerdo estimados por cintilografia de perfusão miocárdica: comparação entre os programas Quantitative Gated SPECT (QGS) e Segami

OBJETIVO: Analisar as diferenças relacionadas ao sexo nas medidas obtidas pelos programas Segami e Quantitative Gated SPECT (QGS). MÉTODOS: Cento e oitenta e um indivíduos assintomáticos sem evidência de cardiopatia foram submetidos a estudos de perfusão miocárdica. O volume diastólico final (VDF), volume sistólico final (VSF) e a fração de ejeção do ventrículo esquerdo (FEVE) foram quantificados pelos programas QGS and Segami para avaliar a influência do sexo, idade, peso, altura, freqüência cardíaca, pressão arterial sistólica, pressão arterial diastólica, índice de massa corporal e área de superfície corporal. RESULTADOS: As médias obtidas com o método QGS foram VDF (mulheres = 68 ml; homens = 95 ml; p < 0,001) e FEVE (mulheres = 66,24%; homens = 58,7%), e com o Segami, VDF (mulheres = 137 ml; homens = 174 ml) e FEVE (mulheres = 62,67%; homens = 58,52%). Foram observadas diferenças significantes entre homens e mulheres no VDF (p < 0,001) e VSF (p < 0,001), que persistiram após o ajuste em relação à área de superfície corporal. CONCLUSÃO: Os volumes ventriculares foram significantemente menores e a FEVE foi significantemente maior em mulheres, de acordo com os programas QGS e Segami.

Valor prognóstico da cintilografia miocárdica de perfusão com tetrofosmin marcado com Tecnécio-99m sincronizada com o ciclo cardíaco (" Gated SPECT" ) na avaliação de pacientes com diabete melito e suspeita clínica de doença arterial coronariana

FUNDAMENTO: A doença cardiovascular é a principal causa de morte em diabéticos, tornando-se primordial a identificação dos indivíduos sob maior risco de eventos cardiovasculares. OBJETIVO: Avaliar o valor prognóstico da cintilografia miocárdica de perfusão com " gated SPECT" em pacientes com diabete melito (DM) e suspeita clínica de doença arterial coronariana. MÉTODOS: Estudo retrospectivo envolvendo 232 pacientes diabéticos submetidos à cintilografia miocárdica com " gated SPECT" . Foram avaliados os parâmetros da cintilografia de perfusão (escores e número de segmentos alterados) e da função ventricular (fração de ejeção, volumes e contratilidade do ventrículo esquerdo). Foram considerados eventos cardiovasculares futuros ocorrência de óbito cardíaco, síndrome coronariana isquêmica aguda, procedimentos de revascularização ou acidente vascular encefálico. Foi realizada a análise uni e multivariada pelo modelo de regressão logística múltipla (p < 0,05). RESULTADOS: Estiveram associados com desfechos futuros na análise univariada: idade (p=0,02); angina de peito (p=0,01); tratamento com insulina (p=0,02); anormalidades na perfusão miocárdica (p<0,0001); número de segmentos envolvidos (p=0,0001); escores de perfusão (p=0,0001); fração de ejeção (p=0,004); volume sistólico final (p=0,03) e achado de alteração segmentar na contratilidade do VE (p<0,0001). Na análise multivariada, o sexo masculino (p=0,007), a idade (p=0,03), a angina (p=0,001), o uso de insulina (p=0,007) e o SDS > 3 (p=0,0001) e o número de segmentos alterados > 3 (p=0,0001) foram preditores de eventos. CONCLUSÃO: A cintilografia miocárdica com " gated SPECT" adiciona informações independentes para a estratificação do risco de eventos cardiovasculares futuros em pacientes com diabete melito e suspeita de doença arterial coronariana.

Fatores biológicos e superestimação da fração de ejeção do ventrículo esquerdo no gated SPECT

FUNDAMENTO: Alguns pacientes apresentam fração de ejeção do ventrículo esquerdo (FEVE) superestimada na cintilografia miocárdica com sincronização eletrocardiográfica (gated SPECT). OBJETIVO: Estabelecer a relação entre fatores biológicos e FEVE superestimada. MÉTODOS: Selecionamos 3.838 pacientes que realizaram gated SPECT entre 20/5/2000 e 16/9/2005, com imagens normais de perfusão e FEVE >50%. Analisamos as variáveis: sexo (29,4% feminino e 70,6% masculino), idade (de 20 a 94 anos - média: 56 anos), peso (de 33,5 a 150 kg - média: 79,6 kg), altura (de 138 a 220 cm - média: 171 cm) e IMC (de 13,9 a 54 - média: 27,2). Em um subgrupo de 1.002 pacientes que realizaram ecocardiograma, incluímos as variáveis diâmetros diastólico (de 36 a 68 mm - média 47,5 mm) e sistólico (de 22 a 41 mm - média 29,8 mm). Dividimos os pacientes em dois grupos: FEVE normal (<80%) e superestimada (>80%). A Razão de Chances (RC) para apresentar FEVE superestimada foi calculada para cada variável por regressão logística. RESULTADOS: Encontramos as seguintes Razões de Chances (p < 0,005): sexo feminino RC = 3,585 (IC95%: 2,745 a 4,683), idade em anos RC = 1,020 (IC95%: 1,011 a 1,029) e altura em cm RC = 0,893 (IC95%: 0,829 a 0,962). O peso e o IMC não se associaram a FEVE significativamente (p>0,2). No subgrupo de 1.002 pacientes, encontramos influência estatisticamente significativa na obtenção da FEVE superestimada para as variáveis diâmetro sistólico, sexo e altura. CONCLUSÃO: Apesar de o diâmetro sistólico influenciar na obtenção da FEVE superestimada, as variáveis sexo e altura apresentam influência independente na superestimação da FEVE pelo gated SPECT.

Função ventricular após cirurgia de revascularização: Gated SPECT comparado à ressonância cardíaca

FUNDAMENTO: A avaliação da função ventricular esquerda pode ser limitada pela movimentação anômala do septo interventricular, freqüentemente encontrada após cirurgia de revascularização miocárdica (CRM). A validação do Gated SPECT como ferramenta para avaliação da função ventricular nesse grupo de pacientes é escassa. OBJETIVO: Investigamos a concordância e a correlação entre a fração de ejeção do ventrículo esquerdo (FEVE), o volume diastólico final (VDF) e o volume sistólico final (VDF), obtidos pela cintilografia de perfusão miocárdica tomográfica sincronizada pelo eletrocardiograma (Gated SPECT), com os mesmos parâmetros medidos pela ressonância magnética cardíaca em 20 pacientes submetidos à revascularização miocárdica. MÉTODOS: A correlação foi medida pelo coeficiente de correlação de Spearman (ρ), enquanto a concordância foi avaliada por meio da análise de Bland e Altman. RESULTADOS: Houve uma correlação boa entre o Gated SPECT e a ressonância magnética cardíaca nos pacientes após CRM em relação à fração de ejeção do ventrículo esquerdo (ρ = 0,85; p = 0,0001), uma correlação moderada para o volume sistólico final (ρ = 0,51; p = 0,02) e uma correlação insignificante para o volume diastólico final (ρ = 0,13; p = 0,5). Os limites de concordância para FEVE, VSF e VDF foram: de -20% a 12%; de -38 a 54ml e de -96 a 100ml, respectivamente. CONCLUSÃO: A fração de ejeção do ventrículo esquerdo obtida pelo Gated SPECT correlaciona-se de modo confiável com a da ressonância magnética em pacientes submetidos à CRM. Os volumes ventriculares, entretanto, não apresentam uma correlação adequada.

Avaliação da perfusão e função miocárdicas em vítimas de escorpionismo utilizando o Gated-SPECT

FUNDAMENTO: O choque cardiogênico e o edema agudo de pulmão são as principais causas de óbito em pacientes com escorpionismo, cujo mecanismo fisiopatológico ainda é controverso. OBJETIVOS: Investigar a correlação entre os distúrbios da perfusão miocárdica e a função contrátil do ventrículo esquerdo, em vítimas de escorpionismo. MÉTODOS: Quinze pacientes submeteram-se à cintilografia de perfusão miocárdica sincronizada com ECG (Gated SPECT), dentro de 72 horas e 15 dias após o acidente escorpiônico. As imagens foram analisadas visualmente por escore semiquantitativo de perfusão (0 = normal, 4 = ausente) e mobilidade (0 = normal, 4 = acinético), utilizando modelo de 17 segmentos. Para cada paciente foram calculados escores somados de perfusão (ESP) e mobilidade (ESM). A fração de ejeção (FEVE) foi calculada por software comercialmente disponível. RESULTADOS: Na avaliação inicial, 12 dos 15 pacientes apresentaram alterações da contratilidade e da perfusão miocárdica. O ESP foi de 12,5 ± 7,3, o ESM de 17,0 ± 12, 8 e a FEVE de 44,6 ± 16,0%. Houve correlação positiva entre o ESP e o ESM (r = 0,68; p = 0,005) e negativa entre o ESP e a FEVE (r = -0,75; p = 0,0021). Os estudos de seguimento mostraram recuperação da contratilidade global (FEVE de 68,9 ± 9,5, p = 0,0002), segmentar (ESM 2,6 ± 3,1, p = 0,0009) e da perfusão (ESP 3,7 ± 3,3, p = 0,0003). A melhora da FEVE correlacionou-se positivamente com a melhora do ESP (r = 0,72; p = 0,0035). CONCLUSÕES: Alterações perfusionais miocárdicas são comuns no envenenamento escorpiônico e correlacionam-se topograficamente com a disfunção contrátil. A recuperação da contratilidade correlaciona-se com a reversibilidade dos defeitos perfusionais. Estes achados sugerem a participação de alterações perfusionais miocárdicas na fisiopatologia desta forma de insuficiência ventricular aguda. (Arq Bras Cardiol 2010;94(4): 444-451)