Acarbose Mitigates Age-Dependent Alterations in Erythrocyte Membrane Transporters During Aging in Rats.

Acarbose (ACA), a well-studied and effective inhibitor of α-amylase and α-glucosidase, is a postprandial-acting antidiabetic medicine. The membrane of the erythrocyte is an excellent tool for analyzing different physiological and biochemical activities since it experiences a range of metabolic alterations throughout aging. It is uncertain if ACA modulates erythrocyte membrane activities in an age-dependent manner. As a result, the current study was conducted to explore the influence of ACA on age-dependent deteriorated functions of transporters/exchangers, disrupted levels of various biomarkers such as lipid hydroperoxides (LHs), protein carbonyl (PCO), sialic acid (SA), total thiol (-SH), and erythrocyte membrane osmotic fragility. In addition to a concurrent increase in Na+/H+ exchanger activity and concentration of LH, PCO, and osmotic fragility, we also detected a considerable decrease in membrane-linked activities of Ca2+-ATPase (PMCA) and Na+/K+-ATPase (NKA), as well as concentrations of SA and -SH in old-aged rats. The aging-induced impairment of the activities of membrane-bound ATPases and the changed levels of redox biomarkers were shown to be effectively restored by ACA treatment.

A missense mutation converts the Na+,K+-ATPase into an ion channel and causes therapy-resistant epilepsy.

The ion pump Na+,K+-ATPase is a critical determinant of neuronal excitability; however, its role in the etiology of diseases of the central nervous system (CNS) is largely unknown. We describe here the molecular phenotype of a Trp931Arg mutation of the Na+,K+-ATPase catalytic α1 subunit in an infant diagnosed with therapy-resistant lethal epilepsy. In addition to the pathological CNS phenotype, we also detected renal wasting of Mg2+. We found that membrane expression of the mutant α1 protein was low, and ion pumping activity was lost. Arginine insertion into membrane proteins can generate water-filled pores in the plasma membrane, and our molecular dynamic (MD) simulations of the principle states of Na+,K+-ATPase transport demonstrated massive water inflow into mutant α1 and destabilization of the ion-binding sites. MD simulations also indicated that a water pathway was created between the mutant arginine residue and the cytoplasm, and analysis of oocytes expressing mutant α1 detected a nonspecific cation current. Finally, neurons expressing mutant α1 were observed to be depolarized compared with neurons expressing wild-type protein, compatible with a lowered threshold for epileptic seizures. The results imply that Na+,K+-ATPase should be considered a neuronal locus minoris resistentia in diseases associated with epilepsy and with loss of plasma membrane integrity.

Na+/K+-ATPase activity is regionally regulated by acute hypoxia in naked mole-rat brain.

Matching ATP supply and demand is key to neuronal hypoxia-tolerance and failure to achieve this balance leads to excitotoxic cell death in most adult mammalian brains. Ion pumping is the most energy-demanding process in the brain and some hypoxia-tolerant vertebrates coordinately down-regulate ion movement across neuronal membranes to reduce the workload of energy-expensive ion pumps, and particularly the Na+/K+-ATPase. Naked mole-rats are among the most hypoxia-tolerant mammals and achieve a hypometabolic state while maintaining brain [ATP] during severe hypoxia; however, whether ionic homeostasis is plastic in naked mole-rat brain is unknown. To examine this question, we exposed animals to 4 h of normoxia or moderate or severe hypoxia (11 or 3% O2, respectively) and measured changes in brain Na+/K+-ATPase activity. We found that 1) whole body metabolic rate decreased ∼25 and 75% in moderate and severe hypoxia, respectively, and 2) Na+/K+-ATPase activity decreased ∼50% in forebrain but increased 2-fold in cerebellum and was unchanged in brainstem. These results indicate that naked mole-rats acutely modulate brain energy demand in a region-specific manner to prioritize energy usage by the cerebellum. This may support exploration, navigation, and escape behaviours, while also enabling ATP savings when encountering hypoxia in nature.

Measuring enzyme activities in crude homogenates: Na+/K+-ATPase as a case study in optimizing assays.

In this review of assays of Na+/K+-ATPase (NKA), we explore the choices made by researchers assaying the enzyme to investigate its role in physiological regulation. We survey NKA structure and function in the context of how it is typically assayed, and how technical choices influence what can be said about the enzyme. In comparing different methods for extraction and assay of NKA, we identified a series of common pitfalls that compromise the veracity of results. We include experimental work to directly demonstrate how choices in detergents, salts and substrates influence NKA activities measured in crude homogenates. Our review of assay approaches integrates what is known from enzymology, biomedical physiology, cell biology and evolutionary biology, offering a more robust method for assaying the enzyme in meaningful ways, identifying caveats and future directions to explore its structure and function. The goal is to provide the sort of background on the enzyme that should be considered in exploring the function of the enzyme in comparative physiology.

A near-infrared light-controlled, ultrasensitive one-step photoelectrochemical detection of dual cell apoptosis indicators in living cancer cells.

Here, a near-infrared (NIR) light-controlled, ultrasensitive one-step photoelectrochemical (PEC) strategy was constructed to simultaneously detect cell apoptosis indicators, phosphatidylserine (Pho) and sodium-potassium adenosine triphosphatase (Sat), on living cancer cells. Using NIR light as excitation, the signal probe methylene blue (Tagkinetic) could be released, leading to a gradually decreased photocurrent signal Ikinetic; meanwhile, the photocurrent Istable of the signal probe carbon quantum dots (Tagstable) remained stable. The simultaneous detection of Pho and Sat could be achieved based on rapid one-step PEC detection under single NIR light with the assistance of a smart signal decryption strategy with Ikinetic and Istable. Importantly, this proposal provides more effective drug candidates with milder pharmaceutical effect but improved safety.

Region- and neuronal-subtype-specific expression of Na,K-ATPase alpha and beta subunit isoforms in the mouse brain.

Na,K-ATPase is a ubiquitous molecule contributing to the asymmetrical distribution of Na+ and K+ ions across the plasma membrane and maintenance of the membrane potential, a prerequisite of neuronal activity. Na,K-ATPase comprises three subunits (α, ß, and FXYD). The α subunit has four isoforms in mice, with three of them (α1, α2, and α3) expressed in the brain. However, the functional and biological significances of the different brain isoforms remain to be fully elucidated. Recent studies have revealed the association of Atp1a3, a gene encoding α3 subunit, with neurological disorders. To map the cellular distributions of the α subunit isoforms and their coexpression patterns, we evaluated the mRNA expression of Atp1a1, Atp1a2, and Atp1a3 by in situ hybridization in the mouse brain. Atp1a1 and Atp1a3 were expressed in neurons, whereas Atp1a2 was almost exclusively expressed in glial cells. Most neurons coexpressed Atp1a1 and Atp1a3, with highly heterogeneous expression levels across the brain regions and neuronal subtypes. We identified parvalbumin (PV)-expressing GABAergic neurons in the hippocampus, somatosensory cortex, and retrosplenial cortex as an example of a neuronal subtype expressing low Atp1a1 and high Atp1a3. The expression of Atp1b isoforms was also heterogeneous across brain regions and cellular subtypes. The PV-expressing neurons expressed a high level of Atp1b1 and a low level of Atp1b2 and Atp1b3. These findings provide basic information on the region- and neuronal-subtype-dependent expression of Na,K-ATPase α and ß subunit isoforms, as well as a rationale for the selective involvement of neurons expressing high levels of Atp1a3 in neurological disorders.

Distribution of aquaporins and sodium transporters in the gastrointestinal tract of a desert hare, Lepus yarkandensis.

Lepus yarkandensis is a desert hare of the Tarim Basin in western China, and it has strong adaptability to arid environments. Aquaporins (AQPs) are a family of water channel proteins that facilitate transmembrane water transport. Gastrointestinal tract AQPs are involved in fluid absorption in the small intestine and colon. This study aimed to determine the distribution of AQPs and sodium transporters in the gastrointestinal tract of L. yarkandensis and to compare the expression of these proteins with that in Oryctolagus cuniculus. Immunohistochemistry was performed to analyse the cellular distribution of these proteins, and the acquired images were analysed with IpWin32 software. Our results revealed that AQP1 was located in the colonic epithelium, central lacteal cells, fundic gland parietal cells, and capillary endothelial cells; AQP3 was located in the colonic epithelium, small intestinal villus epithelium, gastric pit and fundic gland; AQP4 was located in the fundic gland, small intestinal gland and colonic epithelium; and epithelial sodium channel (ENaC) and Na+-K+-ATPase were located in the epithelial cells, respectively. The higher expression levels of AQP1, AQP3, ENaC and Na+-K+-ATPase in the colon of L. yarkandensis compared to those in O. cuniculus suggested that L. yarkandensis has a higher capacity for faecal dehydration.

Assessment of Na+/K+ ATPase Activity in Small Rodent and Human Skeletal Muscle Samples.

INTRODUCTION: In skeletal muscle, the Na/K ATPase (NKA) plays essential roles in processes linked to muscle contraction, fatigue, and energy metabolism; however, very little information exists regarding the regulation of NKA activity. The scarcity of information regarding NKA function in skeletal muscle likely stems from methodological constraints, as NKA contributes minimally to total cellular ATP utilization, and therefore contamination from other ATPases prevents the assessment of NKA activity in muscle homogenates. Here we introduce a method that improves accuracy and feasibility for the determination of NKA activity in small rodent muscle samples (5-10 mg) and in human skeletal muscle. METHODS: Skeletal muscle homogenates from mice (n = 6) and humans (n = 3) were used to measure NKA and sarcoplasmic reticulum Ca ATPase (SERCA) activities with the addition of specific ATPase inhibitors to minimize "background noise." RESULTS: We observed that myosin ATPase activity was the major interfering factor for estimation of NKA activity in skeletal muscle homogenates, as the addition of 25 µM of blebbistatin, a specific myosin ATPase inhibitor, considerably minimized "background noise" (threefold) and enabled the determination of NKA maximal activity with values three times higher than previously reported. The specificity of the assay was demonstrated after the addition of 2 mM ouabain, which completely inhibited NKA. On the other hand, the addition of blebbistatin did not affect the ability to measure SERCA function. The coefficient of variation for NKA and SERCA assays were 6.2% and 4.4%, respectively. CONCLUSION: The present study has improved the methodology to determine NKA activity. We further show the feasibility of measuring NKA and SERCA activities from a common muscle homogenate. This methodology is expected to aid in our long-term understanding of how NKA affects skeletal muscle metabolic homeostasis and contractile function in diverse situations.

MiR-192-5p in the Kidney Protects Against the Development of Hypertension.

MicroRNA miR-192-5p is one of the most abundant microRNAs in the kidney and targets the mRNA for ATP1B1 (ß1 subunit of Na+/K+-ATPase). Na+/K+-ATPase drives renal tubular reabsorption. We hypothesized that miR-192-5p in the kidney would protect against the development of hypertension. We found miR-192-5p levels were significantly lower in kidney biopsy specimens from patients with hypertension (n=8) or hypertensive nephrosclerosis (n=32) compared with levels in controls (n=10). Similarly, Dahl salt-sensitive (SS) rats showed a reduced abundance of miR-192-5p in the renal cortex compared with congenic SS.13BN26 rats that had reduced salt sensitivity (n=9; P<0.05). Treatment with anti-miR-192-5p delivered through renal artery injection in uninephrectomized SS.13BN26 rats exacerbated hypertension significantly. Mean arterial pressure on a 4% NaCl high-salt diet at day 14 post anti-miR-192-5p treatment was 16 mm Hg higher than in rats treated with scrambled anti-miR (n=8 and 6; P<0.05). Similarly, Mir192 knockout mice on the high-salt diet treated with Ang II (angiotensin II) for 14 days exhibited a mean arterial pressure 22 mm Hg higher than wild-type mice (n=9 and 5; P<0.05). Furthermore, protein levels of ATP1B1 were higher in Dahl SS rats than in SS.13BN26 rats. Na+/K+-ATPase activity increased in the renal cortex of SS.13BN26 rats 9 days posttreatment with anti-miR-192-5p compared with that of control anti-miR treated rats. Intrarenal knockdown of ATP1B1 attenuated hypertension in SS.13BN26 rats with intrarenal knockdown of miR-192-5p. In conclusion, miR-192-5p in the kidney protects against the development of hypertension, which is mediated, at least in part, by targeting Atp1b1.

Protectin DX increases alveolar fluid clearance in rats with lipopolysaccharide-induced acute lung injury.

Acute respiratory distress syndrome is a life-threatening critical syndrome resulting largely from the accumulation of and the inability to clear pulmonary edema. Protectin DX, an endogenously produced lipid mediator, is believed to exert anti-inflammatory and pro-resolution effects. Protectin DX (5 µg/kg) was injected i.v. 8 h after LPS (14 mg/kg) administration, and alveolar fluid clearance was measured in live rats (n = 8). In primary rat ATII epithelial cells, protectin DX (3.605 × 10-3 mg/l) was added to the culture medium with LPS for 6 h. Protectin DX improved alveolar fluid clearance (9.65 ± 1.60 vs. 15.85 ± 1.49, p < 0.0001) and decreased pulmonary edema and lung injury in LPS-induced lung injury in rats. Protectin DX markedly regulated alveolar fluid clearance by upregulating sodium channel and Na, K-ATPase protein expression levels in vivo and in vitro. Protectin DX also increased the activity of Na, K-ATPase and upregulated P-Akt via inhibiting Nedd4-2 in vivo. In addition, protectin DX enhanced the subcellular distribution of sodium channels and Na, K-ATPase, which were specifically localized to the apical and basal membranes of primary rat ATII cells. Furthermore, BOC-2, Rp-cAMP, and LY294002 blocked the increased alveolar fluid clearance in response to protectin DX. Protectin DX stimulates alveolar fluid clearance through a mechanism partly dependent on alveolar epithelial sodium channel and Na, K-ATPase activation via the ALX/PI3K/Nedd4-2 signaling pathway.

Influence of the drying method on the bioactive compounds and pharmacological activities of rhubarb.

BACKGROUND: Raw rhubarb samples that have been subjected to different drying procedures will have different therapeutic effects, possibly due to processing-induced variations in the chemical composition. In the present work, the fresh materials were processed by smoking, sun-drying, shade-drying and oven-drying at low, moderate and high temperatures. To facilitate the selection of a suitable drying method for rhubarb, the quality of rhubarb processed under various drying conditions was evaluated based on the simultaneous determination of multiple bioactive constituents in combination with bioactivity assays. RESULTS: The total concentrations of 12 compounds in smoked rhubarb were higher than the concentrations of the same components in raw rhubarb and rhubarb products processed using other drying techniques. Smoked rhubarb was found to substantially inhibit Na+ /K+ -ATPase and thrombin. In addition, higher content of anthraquinones led to higher Na+ /K+ -ATPase inhibitory activities, and higher gallic acid content increased the antithrombin capacity. CONCLUSION: The results confirmed that post-harvest fresh plant materials, especially roots, were still physiologically active organs that could undergo series of anti-dehydration mechanisms, including the production of related secondary metabolites during the early stages of dehydration. Therefore, the proper design of drying processes could enhance the quality of rhubarb as well as other similar medicinal plants. © 2018 Society of Chemical Industry.

Osmoregulatory responses to cadmium in reference and historically metal contaminated Gammarus fossarum (Crustacea, Amphipoda) populations.

In order to better understand the variable sensitivities of crustaceans to metals, we investigated the impact of cadmium exposure in 3 populations of Gammarus fossarum from different rivers of France. The first population lives in a Cd-contaminated river from a geochemical background, while the others inhabit Cd-free sites. Osmoregulation, a relevant biomarker to evaluate crustacean health following metal contamination, was used as a proxy to evaluate the intra- and inter-populationnal sensitivities to Cd. Specimens from each population were experimentally exposed to 9 µg Cd2+/L Cd for 7 days and hemolymph osmolality (HO) was then individually measured. In exposed populations, high inter-individual variations in HO values were noted, resulting in their separation into non-impacted and slightly or highly Cd-impacted (with lower HO) animals. In gills of impacted organisms, deep histopathological alterations and protein overexpression of Na+/K+-ATPase and V-H+-ATPase were observed through histology and immunolocalization, while non-impacted animals showed profiles comparable to controls. Moreover, the osmoregulatory processes in the population living in the Cd-contaminated site were impacted by acute Cd exposure in the laboratory as much as for one of the two populations originating from Cd-free sites. The observed changes did not reveal any obvious adaptive osmoregulatory phenomena at the population scale, but they may be due to differences in fitness between individuals and between populations in relation to the features of their respective environments, unrelated with the presence of the metal.

Ammonia excretion in the marine polychaete Eurythoe complanata (Annelida).

Ammonia is a toxic waste product from protein metabolism and needs to be either converted into less toxic molecules or, in the case of fish and aquatic invertebrates, excreted directly as is. In contrast to fish, very little is known regarding the ammonia excretion mechanism and the participating excretory organs in marine invertebrates. In the current study, ammonia excretion in the marine burrowing polychaete Eurythoe complanata was investigated. As a potential site for excretion, the 100-200 µm long, 30-50 µm wide and up to 25 µm thick dentrically branched, well ventilated and vascularized branchiae (gills) were identified. In comparison to the main body, the branchiae showed considerably higher mRNA expression levels of Na+/K+-ATPase, V-type H+-ATPase, cytoplasmic carbonic anhydrase (CA-2), a Rhesus-like protein, and three different ammonia transporters (AMTs). Experiments on the intact organism revealed that ammonia excretion did not occur via apical ammonia trapping, but was regulated by a basolateral localized V-type H+-ATPase, carbonic anhydrase and intracellular cAMP levels. Interestingly, the V-type H+-ATPase seems to play a role in ammonia retention. A 1 week exposure to 1 mmol l-1 NH4Cl (HEA) did not cause a change in ammonia excretion rates, while the three branchial expressed AMTs showed a tendency to be down-regulated. This indicates a shift of function in the branchial ammonia excretion processes under these conditions.

Aedes aegypti Rhesus glycoproteins contribute to ammonia excretion by larval anal papillae.

In larval Aedes aegypti, transcripts of the Rhesus-like glycoproteins AeRh50-1 and AeRh50-2 have been detected in the anal papillae, sites of ammonia (NH3/NH4+) excretion; however, these putative ammonia transporters have not been previously localized or functionally characterized. In this study, we show that the AeRh50s co-immunolocalize with apical V-type H+-ATPase as well as with basal Na+/K+-ATPase in the epithelium of anal papillae. The double-stranded RNA-mediated knockdown of AeRh50-1 and AeRh50-2 resulted in a significant reduction in AeRh50 protein abundance in the anal papillae, and this was coupled to decreased ammonia excretion. The knockdown of AeRh50-1 resulted in decreased hemolymph [NH4+] and pH whereas knockdown of AeRh50-2 had no effect on these parameters. We conclude that the AeRh50s are important contributors to ammonia excretion at the anal papillae of larval A. aegypti, which may be the basis for their ability to inhabit areas with high ammonia levels.

[Effect of a new ROCK inhibitor thiazovivin on the morphology and function of human corneal endothelial cells].

OBJECTIVE: To evaluate the effect of thiazovivin, a novel ROCK inhibitor, on the morphology and function of human corneal endothelial cells(HCECs). METHODS: The primary HCECs were identified by light microscopy and immunofluorescence staining of neuron-specific enolase. To screen the optimal concentration and action time of thiazovivin for maintaining the morphology and function of primary HCECs, Na (+)/K (+)-ATPase and N-cadherin were chosen as indicators, and the morphology and function of HCECs in various concentrations(0 µmol/L, 2 µmol/L, 4 µmol/L, and 6 µmol/L)for different durations(24 h and 48 h)were examined by immunofluorescence experiments. The effect of thiazovivin on the expression of ROCK was investigated by immunofluorescence and Western blot. RESULTS: The primary HCECs cultured were hexagonal, closely packed, homogeneously and obviously stained by neuron-specific enolase. The immunofluorescence staining of Na(+)/K(+)-ATPase showed that when the primary HCECs cultured with various concentrations of thiazovivin(0, 2, 4, 6 µmol/L)for 24 h, the fluorescence were obvious, and the average absorbance values(A)were 1.27±0.08, 3.72±0.17, 21.07±4.67, 3.69±0.34, respectively. And the immunofluorescence staining of N-cadherin revealed that when the primary HCECs treated with 4 µmol/L thiazovivin for 24 h, the cell boundary was clear and the structure of the cells was intact. While the treating time of thiazovivin(4 µmol/L)on HCECs extended to 48 h, the immunofluorescence staining of Na(+)/K(+)-ATPase and N-cadherin showed that compared to HCECs treated with thiazovivin(4 µmol/L)for 24 h, the fluorescence intensity did not change significantly, but the cells arranged slightly untidy. In addition, the immunofluorescence staining of ROCK was weakened and the expression of ROCK was reduced by thiazovivin. Thiazovivin was effective for protecting the morphology and function of HCECs. An optimal improvement in the morphology, connection and function of HCECs was found when the primary HCECs were cultured with 4 µmol/L thiazovivin for 24 h. Moreover, the expression of ROCK protein could be significantly inhibited by thiazovivin. (Chin J Ophthalmol, 2016, 52: 686-692).

Possible senescence associated change in the predominant a-Na+/K+ ATP-ase isoform in the renal cortex of the rat.

With aging the kidney exhibits progressive deterioration, with a decrease in renal function. Most of the filtered Na+ is actively reabsorbed in the proximal tubules through different transporters located in apical membrane. This process is possible because basolateral Na+/K+-ATP-ase generates electrochemical conditions necessary for energetically favorable Na+ transport. The a-subunit is the catalytic domain of Na+/K+-ATP-ase. There are three isoforms of the a/subunit present in rat kidney. The present study was undertaken to examine the expression pattern of rat a-Na+/K+-ATP-ase during senescence. We tested the impact of aging on mRNA expression of a-Na+/K+-ATP-ase in cortex and medulla of aged Wistar rats. We observed a significant expression decrease in mRNA levels and a possible change of isoform in the cortex of aged animals. These expression changes observed for a subunit could be contributing to affect the renal function in conditions of water and salt stress.

Possible senescence associated change in the predominant &alpha -Na+/K+ ATP-ase isoform in the renal cortex of the rat

With aging the kidney exhibits progressive deterioration, with a decrease in renal function. Most of the filtered Na+ is actively reabsorbed in the proximal tubules through different transporters located in apical membrane. This process is possible because basolateral Na+/K+-ATP-ase generates electrochemical conditions necessary for energetically favorable Na+ transport. The α-subunit is the catalytic domain of Na+/K+-ATP-ase. There are three isoforms of the α/subunit present in rat kidney. The present study was undertaken to examine the expression pattern of rat α-Na+/K+-ATP-ase during senescence. We tested the impact of aging on mRNA expression of α-Na+/K+-ATP-ase in cortex and medulla of aged Wistar rats. We observed a significant expression decrease in mRNA levels and a possible change of isoform in the cortex of aged animals. These expression changes observed for αsubunit could be contributing to affect the renal function in conditions of water and salt stress.

Con el avance de la edad los riñones exhiben un deterioro funcional progresivo con disminución de la función renal. La mayor parte del sodio (Na+) filtrado es reabsorbido activamente en los túbulos proximales a través de diferentes transportadores ubicados en la membrana apical. Este proceso es posible por la existencia de la Na+/K+-ATP-asa basolateral, que genera las condiciones electroquímicas necesarias para que el transporte de Na+ sea energéticamente favorable. La subunidad αde la Na+/K+-ATP-asa es el dominio catalítico de la enzima. Existen tres isoformas de subunidad α, que están presentes en el riñón de la rata. En este trabajo se examinan los patrones de expresión de la α-Na+/K+-ATP-asa durante la senescencia. Se estudió así si el aumento de la edad incidía en la expresión del ARNm de la α-Na+/K+-ATP-asa en corteza y médula renal de ratas Wistar senescentes. Se observó una disminución en la expresión del ARNm de la subunidad αy un posible cambio de isoforma predominante en la corteza de los animales senescentes. Los cambios observados para la expresión de la subunidad αpodrían contribuir a afectar la función renal en condiciones de estrés hídrico y salino.

Electrochemical Platform for the Detection of Transmembrane Proteins Reconstituted into Liposomes.

The development of new methods and strategies for the investigation of membrane proteins is limited by poor solubility of these proteins in an aqueous environment and hindered by a number of other problems linked to the instability of the proteins outside lipid bilayers. Therefore, current research focuses on an analysis of membrane proteins incorporated into model lipid membrane, most frequently liposomes. In this work, we introduce a new electrochemical methodology for the analysis of transmembrane proteins reconstituted into a liposomal system. The proposed analytical approach is based on proteoliposomal sample adsorption on the surface of working electrodes followed by analysis of the anodic and cathodic signals of the reconstituted proteins. It works based on the fact that proteins are electroactive species, in contrast to the lipid components of the membranes under the given experimental conditions. Electroanalytical experiments were performed with two transmembrane proteins; the Na(+)/K(+)ATPase that contains transmembrane as well as large extramembraneous segments and the mitochondrial uncoupling protein 1, which is a transmembrane protein essentially lacking extramembraneous segments. Electrochemical analyses of proteoliposomes were compared with analyses of both proteins solubilized with detergents (C12E8 and octyl-PoE) and supported by the following complementary methods: microscopy techniques, protein activity testing, molecular model visualizations, and immunochemical identification of both proteins. The label-free electrochemical platform presented here enables studies of reconstituted transmembrane proteins at the nanomolar level. Our results may contribute to the development of new electrochemical sensors and microarray systems applicable within the field of poorly water-soluble proteins.

Immunohistochemical and ultrastructural evidence of functional organization along the Corydoras paleatus intestine.

The Neotropical catfish, Corydoras paleatus (Callichthyidae) is a facultative air-breathing teleost that makes use of the caudal portion of the intestine as an accessory air-breathing organ. This portion is highly modified, being well vascularized with capillaries between epithelial cells, which makes it well suited for gas exchange. Instead, the cranial portion is a digestion and absorption site, as it has a typical intestinal epithelium with columnar cells arranged in a single row, villi and less vascularized tunica mucosa. Therefore, the intestine was studied by light and electron microscopy to assess differences between the cranial, middle and caudal portions. To characterize the potential for cell proliferation of this organ, we used anti-proliferating cell nuclear antigen antibody and anti-Na(+) K(+) -ATPase monoclonal antibody to detect the presence of Na(+) /K(+) pump. In C. paleatus it was observed that cell dynamics showed a decreasing gradient of proliferation in cranio-caudal direction. Also, the intestine of this catfish is an important organ in ionoregulation: the basolateral Na(+) /K(+) pump may have an active role, transporting Na(+) out of the cell while helping to maintain the repose potential and to regulate cellular volume.

Characterization of Amino Acid Profile and Enzymatic Activity in Adult Rat Astrocyte Cultures.

Astrocytes are multitasking players in brain complexity, possessing several receptors and mechanisms to detect, participate and modulate neuronal communication. The functionality of astrocytes has been mainly unraveled through the study of primary astrocyte cultures, and recently our research group characterized a model of astrocyte cultures derived from adult Wistar rats. We, herein, aim to characterize other basal functions of these cells to explore the potential of this model for studying the adult brain. To characterize the astrocytic phenotype, we determined the presence of GFAP, GLAST and GLT 1 proteins in cells by immunofluorescence. Next, we determined the concentrations of thirteen amino acids, ATP, ADP, adenosine and calcium in astrocyte cultures, as well as the activities of Na(+)/K(+)-ATPase and acetylcholine esterase. Furthermore, we assessed the presence of the GABA transporter 1 (GAT 1) and cannabinoid receptor 1 (CB 1) in the astrocytes. Cells demonstrated the presence of glutamine, consistent with their role in the glutamate-glutamine cycle, as well as glutamate and D-serine, amino acids classically known to act as gliotransmitters. ATP was produced and released by the cells and ADP was consumed. Calcium levels were in agreement with those reported in the literature, as were the enzymatic activities measured. The presence of GAT 1 was detected, but the presence of CB 1 was not, suggesting a decreased neuroprotective capacity in adult astrocytes under in vitro conditions. Taken together, our results show cellular functionality regarding the astrocytic role in gliotransmission and neurotransmitter management since they are able to produce and release gliotransmitters and to modulate the cholinergic and GABAergic systems.