Separate neuronal and glial Na+,K+-ATPase isoforms regulate glucose utilization in response to membrane depolarization and elevated extracellular potassium.

The role of cell type-specific Na+,K+-ATPase isozymes in function-related glucose metabolism was studied using differentiated rat brain cell aggregate cultures. In mixed neuron-glia cultures, glucose utilization, determined by measuring the rate of radiolabeled 2-deoxyglucose accumulation, was markedly stimulated by the voltage-dependent sodium channel agonist veratridine (0.75 micromol/L), as well as by glutamate (100 micromol/L) and the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) (10 micromol/L). Significant stimulation also was elicited by elevated extracellular potassium (12 mmol/L KCl), which was even more pronounced at 30 mmol/L KCl. In neuron-enriched cultures, a similar stimulation of glucose utilization was obtained with veratridine, specific ionotropic glutamate receptor agonists, and 30 mmol/L but not 12 mmol/L KCl. The effects of veratridine, glutamate, and NMDA were blocked by specific antagonists (tetrodotoxin, CNQX, or MK801, respectively). Low concentrations of ouabain (10(-6) mol/L) prevented stimulation by the depolarizing agents but reduced only partially the response to 12 mmol/L KCl. Together with previous data showing cell type-specific expression of Na+,K+-ATPase subunit isoforms in these cultures, the current results support the view that distinct isoforms of Na+,K+-ATPase regulate glucose utilization in neurons in response to membrane depolarization, and in glial cells in response to elevated extracellular potassium.

Signal Transduction, Plasma Membrane Calcium Movements, and Pigment Translocation in Freshwater Shrimp Chromatophores

Crustacean color change results from the differential translocation of chromatophore pigments, regulated by neurosecretory peptides like red pigment concentrating hormone (RPCH) that, in the red ovarian chromatophores of the freshwater shrimp Macrobrachium olfersi, triggers pigment aggregation via increased cytosolic cGMP and Ca(2+) of both smooth endoplasmatic reticulum (SER) and extracellular origin. However, Ca(2+) movements during RPCH signaling and the mechanisms that regulate intracellular [Ca(2+)] are enigmatic. We investigate Ca(2+) transporters in the chromatophore plasma membrane and Ca(2+) movements that occur during RPCH signal transduction. Inhibition of the plasma membrane Ca(2+)-ATPase by La(3+) and indirect inhibition of the Na(+)/Ca(2+) exchanger by ouabain induce pigment aggregation, revealing a role for both in Ca(2+) extrusion. Ca(2+) channel blockade by La(3+) or Cd(2+) strongly inhibits slow-phase RPCH-triggered aggregation during which pigments disperse spontaneously. L-type Ca(2+) channel blockade by gabapentin markedly reduces rapid-phase translocation velocity; N- or P/Q-type blockade by omega-conotoxin MVIIC strongly inhibits RPCH-triggered aggregation and reduces velocity, effects revealing RPCH-signaled influx of extracellular Ca(2+). Plasma membrane depolarization, induced by increasing external K(+) from 5 to 50 mM, produces Ca(2+)-dependent pigment aggregation, whereas removal of K(+) from the perfusate causes pigment hyperdispersion, disclosing a clear correlation between membrane depolarization and pigment aggregation; K(+) channel blockade by Ba(2+) also partially inhibits RPCH action. We suggest that, during RPCH signal transduction, Ca(2+) released from the SER, together with K(+) channel closure, causes chromatophore membrane depolarization, leading to the opening of predominantly N- and/or P/Q-type voltage-gated Ca(2+) channels, and a Ca(2+)/cGMP cascade, resulting in pigment aggregation. J. Exp. Zool. 313A:605-617, 2010. (C) 2010 Wiley-Liss, Inc.

Membrane properties and synaptic integration of identified neuronal populations in the developing rodent neocortex

In my PhD work I concentrated on three elementary questions that are essential to understand the interactions between the different neuronal cell populations in the developing neocortex. The questions regarded the identity of Cajal-Retzius (CR) cells, the ubiquitous expression of glycine receptors in all major cell populations of the immature neocortex, and the role of taurine in the modulation of immature neocortical network activity.rnTo unravel whether CR cells of different ontogenetic origin have divergent functions I investigated the electrophysiological properties of YFP+ (derived from the septum and borders of the pallium) and YFP− CR cells (derived from other neocortical origins). This study demonstrated that the passive and active electrophysiological properties as well as features of GABAergic PSCs and glutamatergic currents are similar between both CR cell populations. These findings suggest that CR cells of different origins most probably support similar functions within the neuronal networks of the early postnatal cerebral cortex.rnTo elucidate whether glycine receptors are expressed in all major cell populations of the developing neocortex I analyzed the functional expression of glycine receptors on subplate (SP) cells. Activation of glycine receptors by glycine, -alanine and taurine elicited membrane responses that could be blocked by the selective glycinergic antagonist strychnine. Pharmacological experiments suggest that SP cells express functional heteromeric glycine receptors that do not contain 1 subunits. The activation of glycine receptors by glycine and taurine induced a membrane depolarization, which mediated excitatory effects. Considering the key role of SP cells in immature cortical networks and the development of thalamocortical connections, this glycinergic excitation may influence the properties of early cortical networks and the formation of cortical circuits.rnIn the third part of my project I demonstrated that tonic taurine application induced a massive increase in the frequency of PSCs. Based on their reversal potential and their pharmacological properties these taurine-induced PSCs are exclusively transmitted via GABAA receptors to the pyramidal neurons, while both GABAA and glycine receptors were implicated in the generation of the presynaptic activity. Accordingly, whole-cell and cell-attached recordings from genetically labeled interneurons revealed the expression of glycine and GABAA receptors, which mediated an excitatory action on these cells. These findings suggest that low taurine concentrations can tonically activate exclusively GABAergic networks. The activity level maintained by this GABAergic activity in the immature nervous system may contribute to network properties and can facilitate the activity dependent formation of adequate synaptic projections.rnIn summary, the results of my studies complemented the knowledge about neuronal interactions in the immature neocortex and improve our understanding of cellular processes that guide neuronal development and thus shape the brain.rn

Near-membrane [Ca2+] transients resolved using the Ca2+ indicator FFP18.

(Ca2+)-sensitive processes at cell membranes involved in contraction, secretion, and neurotransmitter release are activated in situ or in vitro by Ca2+ concentrations ([Ca2+]) 10-100 times higher than [Ca2+] measured during stimulation in intact cells. This paradox might be explained if the local [Ca2+] at the cell membrane is very different from that in the rest of the cell. Soluble Ca2+ indicators, which indicate spatially averaged cytoplasmic [Ca2+], cannot resolve these localized, near-membrane [Ca2+] signals. FFP18, the newest Ca2+ indicator designed to selectively monitor near-membrane [Ca2+], has a lower Ca2+ affinity and is more water soluble than previously used membrane-associating Ca2+ indicators. Images of the intracellular distribution of FFP18 show that >65% is located on or near the plasma membrane. [Ca2+] transients recorded using FFP18 during membrane depolarization-induced Ca2+ influx show that near-membrane [Ca2+] rises faster and reaches micromolar levels at early times when the cytoplasmic [Ca2+], recorded using fura-2, has risen to only a few hundred nanomolar. High-speed series of digital images of [Ca2+] show that near-membrane [Ca2+], reported by FFP18, rises within 20 msec, peaks at 50-100 msec, and then declines. [Ca2+] reported by fura-2 rose slowly and continuously throughout the time images were acquired. The existence of these large, rapid increases in [Ca2+] directly beneath the surface membrane may explain how numerous (Ca2+)-sensitive membrane processes are activated at times when bulk cytoplasmic [Ca2+] changes are too small to activate them.

Analysis of chemokines and reactive oxygen species formation by rat and human neutrophils induced by microcystin-LA, -YR and -LR

Microcystins (MC), a family of heptapeptide toxins produced by some genera of Cyanobacteria, have potent hepatotoxicity and tumor-promoting activity. Leukocyte infiltration in the liver was observed in MC-induced acute intoxication. Although the mechanisms of hepatotoxicity are still unclear, neutrophil infiltration in the liver may play an important role in triggering toxic injury and tumor development. The present study reports the effects of MC-LA, MC-YR and MC-LR (1 and 1000 nM) on human and rat neutrophils functions in vitro. Cell viability, DNA fragmentation, mitochondrial membrane depolarization and intracellular reactive oxygen species (ROS) levels were measured by flow cytometry. Extracellular ROS content was measured by lucigenin-amplified chemiluminescence, and cytokines were determined by ELISA. We found that these MC increased interleukin-8 (IL-8), cytokine-induced neutrophil chemoattractant-2 alpha beta (CINC-2 alpha beta) and extracellular ROS levels in human and rat neutrophils. Apart from neutrophil presence during the inflammatory process of MC-induced injury, our results suggest that hepatic neutrophil accumulation is further increased by MC-induced neutrophil-derived chemokine. (c) 2008 Elsevier Ltd. All rights reserved.

A new nitrosyl ruthenium complex nitric oxide donor presents higher efficacy than sodium nitroprusside on relaxation of airway smooth muscle

Nitric oxide (NO) has been demonstrated to be the primary agent in relaxing airways in humans and animals. We investigated the mechanisms involved in the relaxation induced by NO-donors, ruthenium complex [Ru(terpy)(bdq)NO(+)](3+) (TERPY) and sodium nitroprusside (SNP) in isolated trachea of rats contracted with carbachol in an isolated organs chamber. For instance, we verified the contribution of K(+) channels, the importance of sGC/cGMP pathway, the influence of the extra and intracellular Ca(2+) sources and the contribution of the epithelium on the relaxing response. Additionally, we have used confocal microscopy in order to analyze the action of the NO-donors on cytosolic Ca(2+) concentration. The results demonstrated that both compounds led to the relaxation of trachea in a dependent-concentration way. However, the maximum effect (E(max)) of TERPY is higher than the SNP. The relaxation induced by SNP (but not TERPY) was significantly reduced by pretreatment with ODQ (sGC inhibitor). Only TERPY-induced relaxation was reduced by tetraethylammonium (K(+) channels blocker) and by pre-contraction with 75 mM KCl (membrane depolarization). The response to both NO-donors was not altered by the presence of thapsigargin (sarcoplasmic reticulum Ca(2+)-ATPase inhibitor). The epithelium removal has reduced the relaxation only to SNP, and it has no effect on TERPY. The both NO-donors reduced the contraction evoked by Ca(2+) influx, while TERPY have shown a higher inhibitory effect on contraction. Moreover, the TERPY was more effective than SNP in reducing the cytosolic Ca(2+) concentration measured by confocal microscopy. In conclusion, these results show that TERPY induces airway smooth muscle relaxation by cGMP-independent mechanisms, it involves the fluxes of Ca(2+) and K(+) across the membrane, it is more effective in reducing cytosolic Ca(2+) concentration and inducing relaxation in the rat trachea than the standard drug, SNP. (C) 2011 Elsevier B.V. All rights reserved.

Effects of exercise on leukocyte death: prevention by hydrolyzed whey protein enriched with glutamine dipeptide

Lymphocyte and neutrophil death induced by exercise and the role of hydrolyzed whey protein enriched with glutamine dipeptide (Gln) supplementation was investigated. Nine triathletes performed two exhaustive exercise trials with a 1-week interval in a randomized, double blind, crossover protocol. Thirty minutes before treadmill exhaustive exercise at variable speeds in an inclination of 1% the subjects ingested 50 g of maltodextrin (placebo) or 50 g of maltodextrin plus 4 tablets of 700 mg of hydrolyzed whey protein enriched with 175 mg of glutamine dipeptide dissolved in 250 mL water. Cell viability, DNA fragmentation, mitochondrial transmembrane potential and production of reactive oxygen species (ROS) were determined in lymphocytes and neutrophils. Exhaustive exercise decreased viable lymphocytes but had no effect on neutrophils. A 2.2-fold increase in the proportion of lymphocytes and neutrophils with depolarized mitochondria was observed after exhaustive exercise. Supplementation of maltodextrin plus Gln (MGln) prevented the loss of lymphocyte membrane integrity and the mitochondrial membrane depolarization induced by exercise. Exercise caused an increase in ROS production by neutrophils, whereas supplementation of MGln had no additional effect. MGln supplementation partially prevented lymphocyte apoptosis induced by exhaustive exercise possibly by a protective effect on mitochondrial function.

Coral snake venoms: mode of action and pathophysiology of experimental envenomation

Coral snakes, the New World Elapidae, are included in the genera Micniroides and Micrurus. The genus Mlcrurus comprises nearly all coral snake species and those which are responsible for human snake-bite accidents. The following generalizations concerning the effects induced by their venoms, and their venom-properties can be made. Coral snake venoms are neurotoxic, producing loss of muscle strenght and death by respiratory paralysis. Local edema and necrosis are not induced nor blood coagulation or hemorrhages. Proteolysis activity is absent or of very low grade. They display phospholipase A2 activity. Nephrotoxic effects are not evoked. The main toxins from elapid venoms are postsynaptic and presynaptic neurotoxins and cardiotoxins. Phospholipases A2 endowed with myonecrotic or cardiotoxin-like properties are important toxic components from some elapid venoms. The mode of action of Micrurus frontalis, M. lemniscatus, M. corallinus and M. fulvius venoms has been investigated in isolated muscle preparations and is here discussed. It is shown that while M. frontalis and M. lemniscatus venoms must contain only neurotoxins that act at the cholinergic end-plate receptor (postsynaptic neurotoxins), M. corallinus venom also inhibits evoked acetylcholine release by the motor nerve endings (presynaptic neurotoxin-like effect) and M. fulvius induces muscle fiber membrane depolarization (cardiotoxin-like effect). The effects produced by M. corallinus and M. fulvius venoms in vivo in dogs and M. frontalis venom in dogs and monkeys are also reported.

Critical role for the lactate transporter, monocarboxylate transporter 1 (mct1), in the regeneration of peripheral nerves

Axons, and particularly regenerating axons, have high metabolic needs in order to maintain critical functions such as axon transport and membrane depolarization. Though some of the required energy likely comes form extracellular glucose and ATP generated in the soma, we and others hypothesize that some of the energy may be supplied by lactate. Unlike glucose that requires glycolytic enzymes to produce pyruvate, lactate can be converted directly to pyruvate by lactate dehydrogenase and transported into mitochondria for oxidative metabolism. In order to be transported into or out of cells, lactate requires specific monocarboxylate transporters (MCTs), the most abundant of which is MCT1. If MCT1 and lactate are critical for nerve function and regeneration, we hypothesize that MCT1 heterozygote null mice, which appear phenotypically normal despite having approximately 40% MCT1 as compared to wildtype littermate mice, would have reduced capacity for repair following nerve injury. To investigate this, adult MCT1 heterozygote null mice or wild-type mice underwent unilateral sciatic nerve crush in the proximal thigh. We found that regeneration of the sciatic nerve, as measured by recovery of compound muscle action potentials (CMAP) in the lateral plantar muscles following proximal sciatic nerve stimulation, was delayed from a median of 21 days in wildtype mice to 38.5 days in MCT1 heterozygote mice. In fact, half of the MCT1 heterozygote null mice had no recovery of CMAP by the endpoint of the study at 42 days, while all of the wild-type mice had recovered. In addition, the maximal amplitude of CMAP recovery in MCT1 heterozygote mull mice was reduced from a mean of 3 mV to 0.5 mV. As would be expected, the denervated gastrocnemius muscle of MCT1 heterozygote null mice remained atrophic at 42 days compared to wild-type mice. Our experiments show that lactate supplied through MCT1 is necessary for nerve regeneration. Experiments are underway to determine whether loss of MCT1 prevents nerve regrowth directly due to reduced energy supply to axons or indirectly by dysfunctional Schwann cells normally dependent on lactate supply through MCT1.

Gluco-incretins regulate beta-cell glucose competence by epigenetic silencing of Fxyd3 expression.

BACKGROUND/AIMS: Gluco-incretin hormones increase the glucose competence of pancreatic beta-cells by incompletely characterized mechanisms. METHODS: We searched for genes that were differentially expressed in islets from control and Glp1r-/-; Gipr-/- (dKO) mice, which show reduced glucose competence. Overexpression and knockdown studies; insulin secretion analysis; analysis of gene expression in islets from control and diabetic mice and humans as well as gene methylation and transcriptional analysis were performed. RESULTS: Fxyd3 was the most up-regulated gene in glucose incompetent islets from dKO mice. When overexpressed in beta-cells Fxyd3 reduced glucose-induced insulin secretion by acting downstream of plasma membrane depolarization and Ca++ influx. Fxyd3 expression was not acutely regulated by cAMP raising agents in either control or dKO adult islets. Instead, expression of Fxyd3 was controlled by methylation of CpGs present in its proximal promoter region. Increased promoter methylation reduced Fxyd3 transcription as assessed by lower abundance of H3K4me3 at the transcriptional start site and in transcription reporter assays. This epigenetic imprinting was initiated perinatally and fully established in adult islets. Glucose incompetent islets from diabetic mice and humans showed increased expression of Fxyd3 and reduced promoter methylation. CONCLUSIONS/INTERPRETATION: Because gluco-incretin secretion depends on feeding the epigenetic regulation of Fxyd3 expression may link nutrition in early life to establishment of adult beta-cell glucose competence; this epigenetic control is, however, lost in diabetes possibly as a result of gluco-incretin resistance and/or de-differentiation of beta-cells that are associated with the development of type 2 diabetes.

Defective TNF-alpha-mediated hepatocellular apoptosis and liver damage in acidic sphingomyelinase knockout mice

This study addressed the contribution of acidic sphingomyelinase (ASMase) in TNF-alpha-mediated hepatocellular apoptosis. Cultured hepatocytes depleted of mitochondrial glutathione (mGSH) became sensitive to TNF-alpha, undergoing a time-dependent apoptotic cell death preceded by mitochondrial membrane depolarization, cytochrome c release, and caspase activation. Cyclosporin A treatment rescued mGSH-depleted hepatocytes from TNF-alpha-induced cell death. In contrast, mGSH-depleted hepatocytes deficient in ASMase were resistant to TNF-alpha-mediated cell death but sensitive to exogenous ASMase. Furthermore, although in vivo administration of TNF-alpha or LPS to galactosamine-pretreated ASMase(+/+) mice caused liver damage, ASMase(-/-) mice exhibited minimal hepatocellular injury. To analyze the requirement of ASMase, we assessed the effect of glucosylceramide synthetase inhibition on TNF-alpha-mediated apoptosis. This approach, which blunted glycosphingolipid generation by TNF-alpha, protected mGSH-depleted ASMase(+/+) hepatocytes from TNF-alpha despite enhancement of TNF-alpha-stimulated ceramide formation. To further test the involvement of glycosphingolipids, we focused on ganglioside GD3 (GD3) because of its emerging role in apoptosis through interaction with mitochondria. Analysis of the cellular redistribution of GD3 by laser scanning confocal microscopy revealed the targeting of GD3 to mitochondria in ASMase(+/+) but not in ASMase(-/-) hepatocytes. However, treatment of ASMase(-/-) hepatocytes with exogenous ASMase induced the colocalization of GD3 and mitochondria. Thus, ASMase contributes to TNF-alpha-induced hepatocellular apoptosis by promoting the mitochondrial targeting of glycosphingolipids.

Gluco-incretins control insulin secretion at multiple levels as revealed in mice lacking GLP-1 and GIP receptors.

The role of the gluco-incretin hormones GIP and GLP-1 in the control of beta cell function was studied by analyzing mice with inactivation of each of these hormone receptor genes, or both. Our results demonstrate that glucose intolerance was additively increased during oral glucose absorption when both receptors were inactivated. After intraperitoneal injections, glucose intolerance was more severe in double- as compared to single-receptor KO mice, and euglycemic clamps revealed normal insulin sensitivity, suggesting a defect in insulin secretion. When assessed in vivo or in perfused pancreas, insulin secretion showed a lack of first phase in Glp-1R(-/-) but not in Gipr(-/-) mice. In perifusion experiments, however, first-phase insulin secretion was present in both types of islets. In double-KO islets, kinetics of insulin secretion was normal, but its amplitude was reduced by about 50% because of a defect distal to plasma membrane depolarization. Thus, gluco-incretin hormones control insulin secretion (a) by an acute insulinotropic effect on beta cells after oral glucose absorption (b) through the regulation, by GLP-1, of in vivo first-phase insulin secretion, probably by an action on extra-islet glucose sensors, and (c) by preserving the function of the secretory pathway, as evidenced by a beta cell autonomous secretion defect when both receptors are inactivated.

Intracellular ATP Decrease Mediates NLRP3 Inflammasome Activation upon Nigericin and Crystal Stimulation.

Activation of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome initiates an inflammatory response, which is associated with host defense against pathogens and the progression of chronic inflammatory diseases such as gout and atherosclerosis. The NLRP3 inflammasome mediates caspase-1 activation and subsequent IL-1β processing in response to various stimuli, including extracellular ATP, although the roles of intracellular ATP (iATP) in NLRP3 activation remain unclear. In this study, we found that in activated macrophages artificial reduction of iATP by 2-deoxyglucose, a glycolysis inhibitor, caused mitochondrial membrane depolarization, leading to IL-1β secretion via NLRP3 and caspase-1 activation. Additionally, the NLRP3 activators nigericin and monosodium urate crystals lowered iATP through K(+)- and Ca(2+)-mediated mitochondrial dysfunction, suggesting a feedback loop between iATP loss and lowering of mitochondrial membrane potential. These results demonstrate the fundamental roles of iATP in the maintenance of mitochondrial function and regulation of IL-1β secretion, and they suggest that maintenance of the intracellular ATP pools could be a strategy for countering NLRP3-mediated inflammation.

Cardiac tissue engineering by electrical stimulation with subcutaneous and cardiac adipose-tissue derived progenitor cells (ATDPCs)

A major challenge of cardiac tissue engineering is directing cells to establish the physiological structure and function of the myocardium being replaced. In native heart, pacing cells generate electrical stimuli that spread throughout the heartcausing cell membrane depolarization and activation of contractile apparatus. We ought to examine whether electricalstimulation of adipose tissue-derived progenitor cells (ATDPCs) exerts phenotypic and genetic changes that enhance theircardiomyogenic potential.

Outward potassium current oscillations in macrophage polykaryons: extracellular calcium entry and calcium-induced calcium release

Outward current oscillations associated with transient membrane hyperpolarizations were induced in murine macrophage polykaryons by membrane depolarization in the absence of external Na+. Oscillations corresponded to a cyclic activation of Ca2+-dependent K+ currents (IKCa) probably correlated with variations in intracellular Ca2+ concentration. Addition of external Na+ (8 mM) immediately abolished the outward current oscillations, suggesting that the absence of the cation is necessary not only for their induction but also for their maintenance. Oscillations were completely blocked by nisoldipine. Ruthenium red and ryanodine reduced the number of outward current cycles in each episode, whereas quercetin prolonged the hyperpolarization 2- to 15-fold. Neither low molecular weight heparin nor the absence of a Na+ gradient across the membrane had any influence on oscillations. The evidence suggests that Ca2+ entry through a pathway sensitive to Ca2+ channel blockers is elicited by membrane depolarization in Na+-free medium and is essential to initiate oscillations, which are also dependent on the cyclic release of Ca2+ from intracellular Ca2+-sensitive stores; Ca2+ ATPase acts by reducing intracellular Ca2+, thus allowing slow deactivation of IKCa. Evidence is presented that neither a Na+/Ca2+ antiporter nor Ca2+ release from IP3-sensitive Ca2+ stores participate directly in the mechanism of oscillation