Erythrocytes may contain a ouabain-insensitive K+-ATPase which plays a role in internal K+ balance

Erythrocytes are useful in evaluating K+ transport pathways involved in internal K+ balance. Several forms of H+,K+-ATPase have been described in nephron segments active in K+ transport. Furthermore, the activity of a ouabain-insensitive isoform of H+,K+-ATPase expressed in collecting duct cells may be modulated by acid-base status. Various assays were performed to determine if a ouabain-insensitive K+-ATPase is present in rat erythrocytes and, if so, whether it plays a role in internal K+ balance. Kinetic studies demonstrated that maximal stimulation of enzyme activity was achieved with 2.5 mM K+ at pH 7.4. Subsequent experiments were performed on erythrocyte membranes collected from animals submitted to varying degrees of K+ homeostasis: control rats, K+-depleted rats, K+-loaded rats, and rats rendered hyperkalemic due to acute renal failure. As observed in the collecting duct cell studies, there was a significant decrease in the activity of ouabain-insensitive K+-ATPase in the erythrocytes of both K+-loaded and metabolically alkalotic K+-depleted rats. However, this enzyme activity in erythrocyte membranes of rats with metabolic acidosis-related hyperkalemia was similar to that of control animals. This finding may be interpreted as resulting from two potentially modulating factors: the stimulating effect that metabolic acidosis has on K+-ATPase and the counteracting effect that hyperkalemia and uremia have on metabolic acidosis. In summary, we present evidence of a ouabain-insensitive K+-ATPase in erythrocytes, whose activity is modulated by acid-base status and K+ levels.

The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells

8-Methoxy psoralen (8-MOP) exerts a short-term (24 h) mitogenic action, and a long-term (48-72 h) anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 µM). The intracellular Ca2+ chelator BAPTA/AM (1 µM) blocked both early (mitogenic) and late (anti-proliferative and melanogenic) 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels) did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 µM protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92% increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.

Participation of ATP-sensitive K+ channels in the peripheral antinociceptive effect of fentanyl in rats

We examined the effect of several K+ channel blockers such as glibenclamide, tolbutamide, charybdotoxin (ChTX), apamin, tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP), and cesium on the ability of fentanyl, a clinically used selective µ-opioid receptor agonist, to promote peripheral antinociception. Antinociception was measured by the paw pressure test in male Wistar rats weighing 180-250 g (N = 5 animals per group). Carrageenan (250 µg/paw) decreased the threshold of responsiveness to noxious pressure (delta = 188.1 ± 5.3 g). This mechanical hyperalgesia was reduced by fentanyl (0.5, 1.5 and 3 µg/paw) in a peripherally mediated and dose-dependent fashion (17.3, 45.3 and 62.6%, respectively). The selective blockers of ATP-sensitive K+ channels glibenclamide (40, 80 and 160 µg/paw) and tolbutamide (80, 160 and 240 µg/paw) dose dependently antagonized the antinociception induced by fentanyl (1.5 µg/paw). In contrast, the effect of fentanyl was unaffected by the large conductance Ca2+-activated K+ channel blocker ChTX (2 µg/paw), the small conductance Ca2+-activated K+ channel blocker apamin (10 µg/paw), or the non-specific K+ channel blocker TEA (150 µg/paw), 4-AP (50 µg/paw), and cesium (250 µg/paw). These results extend previously reported data on the peripheral analgesic effect of morphine and fentanyl, suggesting for the first time that the peripheral µ-opioid receptor-mediated antinociceptive effect of fentanyl depends on activation of ATP-sensitive, but not other, K+ channels.

Mitochondrial K+ transport and cardiac protection during ischemia/reperfusion

Mitochondrial ion transport, oxidative phosphorylation, redox balance, and physical integrity are key factors in tissue survival following potentially damaging conditions such as ischemia/reperfusion. Recent research has demonstrated that pharmacologically activated inner mitochondrial membrane ATP-sensitive K+ channels (mitoK ATP) are strongly cardioprotective under these conditions. Furthermore, mitoK ATP are physiologically activated during ischemic preconditioning, a procedure which protects against ischemic damage. In this review, we discuss mechanisms by which mitoK ATP may be activated during preconditioning and the mitochondrial and cellular consequences of this activation, focusing on end-effects which may promote ischemic protection. These effects include decreased loss of tissue ATP through reverse activity of ATP synthase due to increased mitochondrial matrix volumes and lower transport of adenine nucleotides into the matrix. MitoK ATP also decreases the release of mitochondrial reactive oxygen species by promoting mild uncoupling in concert with K+/H+ exchange. Finally, mitoK ATP activity may inhibit mitochondrial Ca2+ uptake during ischemia, which, together with decreased reactive oxygen release, can prevent mitochondrial permeability transition, loss of organelle function, and loss of physical integrity. We discuss how mitochondrial redox status, K+ transport, Ca2+ transport, and permeability transitions are interrelated during ischemia/reperfusion and are determinant factors regarding the extent of tissue damage.

Some physico-chemical parameters that influence proteinase K resistance and the infectivity of PrP Sc after high pressure treatment

Crude brain homogenates of terminally diseased hamsters infected with the 263 K strain of scrapie (PrP Sc) were heated and/or pressurized at 800 MPa at 60ºC for different times (a few seconds or 5, 30, 120 min) in phosphate-buffered saline (PBS) of different pH and concentration. Prion proteins were analyzed on immunoblots for their proteinase K (PK) resistance, and in hamster bioassays for their infectivity. Samples pressurized under initially neutral conditions and containing native PrP Sc were negative on immunoblots after PK treatment, and a 6-7 log reduction of infectious units per gram was found when the samples were pressurized in PBS of pH 7.4 for 2 h. A pressure-induced change in the protein conformation of native PrP Sc may lead to less PK resistant and less infectious prions. However, opposite results were obtained after pressurizing native infectious prions at slightly acidic pH and in PBS of higher concentration. In this case an extensive fraction of native PrP Sc remained PK resistant after pressure treatment, indicating a protective effect possibly due to induced aggregation of prion proteins in such buffers.

Ventricular performance and Na+-K+ ATPase activity are reduced early and late after myocardial infarction in rats

Myocardial infarction leads to compensatory ventricular remodeling. Disturbances in myocardial contractility depend on the active transport of Ca2+ and Na+, which are regulated by Na+-K+ ATPase. Inappropriate regulation of Na+-K+ ATPase activity leads to excessive loss of K+ and gain of Na+ by the cell. We determined the participation of Na+-K+ ATPase in ventricular performance early and late after myocardial infarction. Wistar rats (8-10 per group) underwent left coronary artery ligation (infarcted, Inf) or sham-operation (Sham). Ventricular performance was measured at 3 and 30 days after surgery using the Langendorff technique. Left ventricular systolic pressure was obtained under different ventricular diastolic pressures and increased extracellular Ca2+ concentrations (Ca2+e) and after low and high ouabain concentrations. The baseline coronary perfusion pressure increased 3 days after myocardial infarction and normalized by 30 days (Sham 3 = 88 ± 6; Inf 3 = 130 ± 9; Inf 30 = 92 ± 7 mmHg; P < 0.05). The inotropic response to Ca2+e and ouabain was reduced at 3 and 30 days after myocardial infarction (Ca2+ = 1.25 mM; Sham 3 = 70 ± 3; Inf 3 = 45 ± 2; Inf 30 = 29 ± 3 mmHg; P < 0.05), while the Frank-Starling mechanism was preserved. At 3 and 30 days after myocardial infarction, ventricular Na+-K+ ATPase activity and contractility were reduced. This Na+-K+ ATPase hypoactivity may modify the Na+, K+ and Ca2+ transport across the sarcolemma resulting in ventricular dysfunction.

A key role for Na+/K+-ATPase in the endothelium-dependent oscillatory activity of mouse small mesenteric arteries

Oscillatory contractile activity is an inherent property of blood vessels. Various cellular mechanisms have been proposed to contribute to oscillatory activity. Mouse small mesenteric arteries display a unique low frequency contractile oscillatory activity (1 cycle every 10-12 min) upon phenylephrine stimulation. Our objective was to identify mechanisms involved in this peculiar oscillatory activity. First-order mesenteric arteries were mounted in tissue baths for isometric force measurement. The oscillatory activity was observed only in vessels with endothelium, but it was not blocked by L-NAME (100 µM) or indomethacin (10 µM), ruling out the participation of nitric oxide and prostacyclin, respectively, in this phenomenon. Oscillatory activity was not observed in vessels contracted with K+ (90 mM) or after stimulation with phenylephrine plus 10 mM K+. Ouabain (1 to 10 µM, an Na+/K+-ATPase inhibitor), but not K+ channel antagonists [tetraethylammonium (100 µM, a nonselective K+ channel blocker), Tram-34 (10 µM, blocker of intermediate conductance K+ channels) or UCL-1684 (0.1 µM, a small conductance K+ channel blocker)], inhibited the oscillatory activity. The contractile activity was also abolished when experiments were performed at 20°C or in K+-free medium. Taken together, these results demonstrate that Na+/K+-ATPase is a potential source of these oscillations. The presence of α-1 and α-2 Na+/K+-ATPase isoforms was confirmed in murine mesenteric arteries by Western blot. Chronic infusion of mice with ouabain did not abolish oscillatory contraction, but up-regulated vascular Na+/K+-ATPase expression and increased blood pressure. Together, these observations suggest that the Na+/K+ pump plays a major role in the oscillatory activity of murine small mesenteric arteries.

Participation of the NO/cGMP/K+ATP pathway in the antinociception induced by Walker tumor bearing in rats

Implantation of Walker 256 tumor decreases acute systemic inflammation in rats. Inflammatory hyperalgesia is one of the most important events of acute inflammation. The L-arginine/NO/cGMP/K+ATP pathway has been proposed as the mechanism of peripheral antinociception mediated by several drugs and physical exercise. The objective of this study was to investigate a possible involvement of the NO/cGMP/K+ATP pathway in antinociception induced in Walker 256 tumor-bearing male Wistar rats (180-220 g). The groups consisted of 5-6 animals. Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. Walker tumor (4th and 7th day post-implantation) reduced prostaglandin E2- (PGE2, 400 ng/paw; 50 µL; intraplantar injection) and carrageenan-induced hypernociception (500 µg/paw; 100 µL; intraplantar injection). Walker tumor-induced analgesia was reversed (99.3% for carrageenan and 77.2% for PGE2) by a selective inhibitor of nitric oxide synthase (L-NAME; 90 mg/kg, ip) and L-arginine (200 mg/kg, ip), which prevented (80% for carrageenan and 65% for PGE2) the effect of L-NAME. Treatment with the soluble guanylyl cyclase inhibitor ODQ (100% for carrageenan and 95% for PGE2; 8 µg/paw) and the ATP-sensitive K+ channel (KATP) blocker glibenclamide (87.5% for carrageenan and 100% for PGE2; 160 µg/paw) reversed the antinociceptive effect of tumor bearing in a statistically significant manner (P < 0.05). The present study confirmed an intrinsic peripheral antinociceptive effect of Walker tumor bearing in rats. This antinociceptive effect seemed to be mediated by activation of the NO/cGMP pathway followed by the opening of KATP channels.

Spectrum of K ras mutations in Pakistani colorectal cancer patients

The incidence of colorectal cancer (CRC) is increasing daily worldwide. Although different aspects of CRC have been studied in other parts of the world, relatively little or almost no information is available in Pakistan about different aspects of this disease at the molecular level. The present study was aimed at determining the frequency and prevalence of K ras gene mutations in Pakistani CRC patients. Tissue and blood samples of 150 CRC patients (64% male and 36% female) were used for PCR amplification of K ras and detection of mutations by denaturing gradient gel electrophoresis, restriction fragment length polymorphism analysis, and nucleotide sequencing. The K ras mutation frequency was found to be 13%, and the most prevalent mutations were found at codons 12 and 13. A novel mutation was also found at codon 31. The dominant mutation observed was a G to A transition. Female patients were more susceptible to K ras mutations, and these mutations were predominant in patients with a nonmetastatic stage of CRC. No significant differences in the prevalence of K ras mutations were observed for patient age, gender, or tumor type. It can be inferred from this study that Pakistani CRC patients have a lower frequency of K ras mutations compared to those observed in other parts of the world, and that K ras mutations seemed to be significantly associated with female patients.

Estudo químico de alimentos formulados à base de palmito Bactris gasipaes H.B.K. (pupunha) desidratado

O coração da palmeira Bactris gasipaes H.B.K. (pupunha) foi utilizado, sob forma desidratada, na formulação de uma sopa-creme para merenda escolar. Análises químicas do palmito e do coração da palmeira (açúcares redutores, lipídeos, proteínas totais, aminograma, fibras, calorias e minerais) foram feitas tanto na forma in natura como na forma processada, bem como na de sopa-creme. Não foram encontradas diferenças relevantes em relação a essas determinações, podendo-se ressaltar a boa qualidade aminoacídica do coração da palmeira.

Características sensoriais de compotas de pêssego light elaboradas com sucralose e acesulfame-K

O trabalho objetivou realizar uma caracterização sensorial de compotas de pêssego com reduzidas calorias. As compotas foram elaboradas pela substituição parcial de açúcar pelos edulcorantes sucralose, acesulfame-K e sucralose+acesulfame-K. Além desta caracterização, investigou-se a vida-de-prateleira destes produtos durante um período de 90 dias. Para isto, foram avaliados os atributos de aparência geral, cor, brilho, maciez, sabor geral e acidez, logo após o processamento, aos 30, 60 e 90 dias de estocagem. Ao término do período de estocagem executou-se, ainda, um teste de preferência. De acordo com a avaliação sensorial dos diferentes atributos, detectou-se diferenças significativas entre as quatro formulações de compota, especialmente em relação aos atributos de sabor e acidez. As formulações com acesulfame-K e sucralose+acesulfame-K mostraram valores inferiores às demais. Durante o período de armazenamento não se constatou expressivas alterações nas características sensorias das compotas. Os resultados do teste de preferência mostraram que não houve distinção na preferência dos consumidores por qualquer das formulações.

Estabilidade de ácido ascórbico e antocianinas em néctar de camu-camu (Myrciaria dubia (H. B. K.) McVaugh)

O camu-camu é um fruto com notável potencial nutricional, pelo seu alto conteúdo em ácido ascórbico. Sendo assim, objetivou-se avaliar a estabilidade do ácido ascórbico e pigmentos presentes no néctar de camu-camu, armazenados sob diferentes condições de luminosidade e temperatura. Os frutos foram despolpados e avaliados quanto às características físico-químicas. O néctar, obtido a partir da polpa, foi acondicionado em garrafas PET e armazenado em temperatura ambiente e sob refrigeração na presença e ausência de luz, sendo avaliado por 120 dias quanto à estabilidade de ácido ascórbico e antocianinas. O teor de ácido ascórbico nos néctares armazenados sob luz não diferiu estatisticamente dos armazenados protegidos da luz (343,25 e 340,48 mg.100 g -1), respectivamente, nos armazenados sob refrigeração, e (330,48 e 333,56 mg.100 g -1) nos armazenados em temperatura ambiente. Constatou-se que esta vitamina em néctares armazenados por 120 dias em temperatura de refrigeração apresentou boa estabilidade, com perda de apenas 12 a 14%. Quanto às antocianinas, a temperatura ambiente contribuiu negativamente, ocasionando uma degradação mais acelerada, no entanto, a exposição à luz não teve efeito. Nestas condições experimentais, conclui-se que o fator luminosidade tem pouca influência sobre o ácido ascórbico e antocianinas no néctar de camu-camu, e que a temperatura ambiente de armazenamento é fator negativo na estabilidade destes pigmentos.

Propriedades funcionais das proteínas de amêndoas da faveleira (Cnidosculus phyllacanthus (Mart.) Pax. et K. Hoffm.) com e sem espinhos

A faveleira possui amêndoas com potencial em óleos e proteínas alimentares e é encontrada em todos os estados do nordeste brasileiro, principalmente nas regiões do sertão e caatinga. Objetivando a sua aplicação em formulações alimentícias, as propriedades funcionais das proteínas de amêndoas da faveleira com espinhos (FCE), e sem espinhos (FSE) foram estudadas. As amêndoas apresentaram, elevado conteúdo de lipídio (40,56 e 40,21%) e proteína (33,00 e 35,77%) e depois de desengordurada, apresentou 57,55 e 63,00% respectivamente. Na obtenção dos isolados proteicos, os índices em proteínas extraídas foram de 83,51% para a FCE e 80,57% para a FSE em pH 10,5. Os índices de proteínas recuperadas através da precipitação isoelétrica foram de 72,84 e 72,11% em pH 4,5. Os índices de proteínas solúveis e as propriedades de emulsificação mostraram-se dependentes do pH, com valores mínimos no ponto isoelétrico e mais elevados em pH ácido e básico ao pI. O isolado proteico da FSE apresentou melhor capacidade de absorção de água e de óleo em relação à FCE. Quando avaliadas através da atividade e da estabilidade de emulsão os isolados apresentaram performance apropriada tanto na região ácida quanto alcalina do pI.

Association between aflatoxin and aflatoxigenic fungi in Brazil nut (Bertholletia excelsa H.B.K.)

Brazil nut has a high nutritional content and is a very important trade commodity to some Latin American countries. In order to evaluate its safety, 120 samples from different stages of the productive chain were analyzed in terms of: moisture content (mc), aflatoxigenic fungi and aflatoxin (LOQ = 1.95 μg.kg-1 total aflatoxin) using TLC. Among all samples, 4 (6.7%) from the receiving area, and 5 (16.7%), from retail presented aflatoxins above the LOQ, but the amount of aflatoxins was below the LOQ after the samples were dried in the plant. The positive samples were above the limit of total aflatoxin permitted by the European Union (4.0 μg.kg-1) and Brazil (30 μg.kg-1). The mc mean was 22.43% in the receiving area, which is higher than that in the other stages samples. All the A. flavus strains were aflatoxigenic, and there was statistic association between the presence of aflatoxin and flavus strains. The aflatoxigenic fungi strains associated to the aflatoxins levels in the samples show that an effective control is necessary for the food safety in the Brazil nut production chain.