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Maximal muscular power production is of fundamental importance to human functional capacity and feats of performance. Here, we present a synthesis of literature pertaining to physiological systems that limit maximal muscular power during cyclic actions characteristic of locomotor behaviours, and how they adapt to training. Maximal, cyclic muscular power is known to be the main determinant of sprint cycling performance, and therefore we present this synthesis in the context of sprint cycling. Cyclical power is interactively constrained by force-velocity properties (i.e. maximum force and maximum shortening velocity), activation-relaxation kinetics and muscle coordination across the continuum of cycle frequencies, with the relative influence of each factor being frequency dependent. Muscle cross-sectional area and fibre composition appear to be the most prominent properties influencing maximal muscular power and the power-frequency relationship. Due to the role of muscle fibre composition in determining maximum shortening velocity and activation-relaxation kinetics, it remains unclear how improvable these properties are with training. Increases in maximal muscular power may therefore arise primarily from improvements in maximum force production and neuromuscular coordination via appropriate training. Because maximal efforts may need to be sustained for ~15-60 s within sprint cycling competition, the ability to attenuate fatigue-related power loss is also critical to performance. Within this context, the fatigued state is characterised by impairments in force-velocity properties and activation-relaxation kinetics. A suppression and leftward shift of the power-frequency relationship is subsequently observed. It is not clear if rates of power loss can be improved with training, even in the presence adaptations associated with fatigue-resistance. Increasing maximum power may be most efficacious for improving sustained power during brief maximal efforts, although the inclusion of sprint interval training likely remains beneficial. Therefore, evidence from sprint cycling indicates that brief maximal muscular power production under cyclical conditions can be readily improved via appropriate training, with direct implications for sprint cycling as well as other athletic and health-related pursuits. [ABSTRACT FROM AUTHOR]

Innate immune cells, such as macrophages, respond to pathogen-associated molecular patterns, such as a lipopolysaccharide (LPS), to secrete various inflammatory mediators. Recent studies have suggested that damage-associated molecular patterns (DAMPs), released extracellularly from damaged or immune cells, also play a role in the activation of inflammatory responses. In this study, to prevent excess inflammation, we focused on DAMPs-mediated signaling that promotes LPS-stimulated inflammatory responses, especially adenosine 5’-triphosphate (ATP)-triggered signaling through the ionotropic purinergic receptor 7 (P2X7R), as a potential new anti-inflammatory target of natural polyphenols. We focused on the phenomenon that ATP accelerates the production of inflammatory mediators, such as nitric oxide, in LPS-stimulated J774.1 mouse macrophages. Using an siRNA-mediated knockdown and specific antagonist, it was found that the ATP-induced enhanced inflammatory responses were mediated through P2X7R. We then screened 42 polyphenols for inhibiting the ATP/P2X7R-induced calcium influx, and found that several polyphenols exhibited significant inhibitory effects. Especially, a flavonoid baicalein significantly inhibited ATP-induced inflammation, including interleukin-1β secretion, through inhibition of the ATP/P2X7R signaling. These findings suggest that ATP/P2X7R signaling plays an important role in excess inflammatory responses and could be a potential anti-inflammatory target of natural polyphenolic compounds. [ABSTRACT FROM AUTHOR]

Blood levels of the acute phase reactant C-reactive protein (CRP) are frequently measured as a clinical marker for inflammation, but the biological functions of CRP are still controversial. CRP is a phosphocholine (PC)-binding pentraxin, mainly produced in the liver in response to elevated levels of interleukin-1β (IL-1β) and of the IL-1β-dependent cytokine IL-6. While both cytokines play important roles in host defense, excessive systemic IL-1β levels can cause life-threatening diseases such as trauma-associated systemic inflammation. We hypothesized that CRP acts as a negative feedback regulator of monocytic IL-1β maturation and secretion. Here, we demonstrate that CRP, in association with PC, efficiently reduces ATP-induced inflammasome activation and IL-1β release from human peripheral blood mononuclear leukocytes and monocytic U937 cells. Effective concentrations are in the range of marginally pathologic CRP levels (IC50 = 4.9 µg/ml). CRP elicits metabotropic functions at nicotinic acetylcholine (ACh) receptors (nAChRs) containing subunits α7, α9, and α10 and suppresses the function of ATP-sensitive P2X7 receptors in monocytic cells. Of note, CRP does not induce ion currents at conventional nAChRs, suggesting that CRP is a potent nicotinic agonist controlling innate immunity without entailing the risk of adverse effects in the nervous system. In a prospective study on multiple trauma patients, IL-1β plasma concentrations negatively correlated with preceding CRP levels, whereas inflammasome-independent cytokines IL-6, IL-18, and TNF-α positively correlated. In conclusion, PC-laden CRP is an unconventional nicotinic agonist that potently inhibits ATP-induced inflammasome activation and might protect against trauma-associated sterile inflammation. [ABSTRACT FROM AUTHOR]

Abstract: AMP‐activated protein kinase (AMPK) is a conserved sensor of cellular energy change and is activated by increased AMP/ATP and/or ADP/ATP ratios. AMPK maintains the energy balance by decreasing the ATP‐consuming processes such as transcription of synthetic fat genes and rRNA, the translation of ribosomal proteins, synthesis of cholesterol and fatty acid, while the metabolic pathways such as glucose and fatty transport, fatty acid oxidation, autophagy, mitochondrial synthesis and oxidative metabolism are increased to preserve ATP during energy deficiency. Recent advance has demonstrated that AMPK activity has a close association with the initiation and progression in various cancers. Here we review the mechanisms that AMPK controls energy metabolism through regulating ATP synthesis and consumption, and further discuss the deregulation of AMPK in cancers. [ABSTRACT FROM AUTHOR]

Objective Potassium channels of the ATP-sensitive family (K ATP channel) are inhibited by increase in intracellular ATP. Electrophysiological studies have demonstrated that the kinetics and pharmacological properties of K ATP channels vary among different tissues, suggesting structurally and functionally distinct types. There are studies showing human periodontal ligament (PDL) cells respond to mechanical stress by increasing ATP release, which participates in bone resorption or bone homeostasis. So, in this study we investigated the existence of K ATP channel subunit and their single channel properties in human periodontal ligaments. Materials & method The human PDL cells were isolated from healthy erupted third molar. For patch-clamp experiments, human PDL fibroblasts were seeded on 3.5 cm plastic dishes. The inside-out patch clamp recordings were performed under voltage clamp mode. Reverse transcriptase polymerase chain reaction (RT-PCR) was conducted to identify the channel subunits. All pair-wise comparisons were performed by Paired t- test. A P value < 0.05 was considered significant. Results We observed mRNA transcripts for Kir6.1, Kir6.2 and Sur2B subuits in the human PDL cells. In inside-out patch mode, the single channel conductance was 163 pS at symmetrical K + concentration of 140 mM and inward rectification was seen in ATP-free bath solution. The reversal potential of the currents was found to be 0 mV at symmetrical concentration (140 mM) of K + in bath solution. The single channel currents were almost blocked by adding 5 mM ATP in the bath solution. However, the currents were not blocked by 100 μM glibenclamide, a subunit specific K ATP channel blocker. Conclusions These results indicate that human PDL cells express K ATP channels subunit including Sur2B and Kir6.1 and Kir6.2 which are sensitive to ATP but insensitive to glibenclamide. [ABSTRACT FROM AUTHOR]

We investigated the effect of chrysin on isolated normal and chronic lymphocytic leukemia (CLL) B-lymphocytes and their isolated mitochondria. We report that a selective and significant increase in cytotoxicity, intracellular reactive oxygen species, mitochondrial membrane potential collapse, ADP/ATP ratio, caspase 3 activation and finally apoptosis in chrysin-treated CLL B- lymphocytes. Also we determined that chrysin selectively inhibits complex II and ATPases in cancerous mitochondria. In this study we proved that the ability of chrysin to promote apoptosis in CLL B-lymphocytes performed by selectively targeting of mitochondria. Our findings may provide a potential therapeutic approach for using chrysin to target mitochondria in CLL B-lymphocytes. [ABSTRACT FROM PUBLISHER]

Background: Deficits in mitochondrial function and oxidative stress play pivotal roles in Down syndrome (DS) and Alzheimer's disease (AD) and these alterations in mitochondria occur systemically in both conditions.Objective: We hypothesized that peripheral cells of elder subjects with DS exhibit disease-specific and dementia-specific metabolic features. To test this, we performed a comprehensive analysis of energy metabolism in lymphoblastic-cell-lines (LCLs) derived from subjects belonging to four groups: DS-with-dementia (DSAD), DS-without-dementia (DS), sporadic AD, and age-matched controls.Methods: LCLs were studied under regular or minimal feeding regimes with galactose or glucose as primary carbohydrate sources. We assessed metabolism under glycolysis or oxidative phosphorylation by quantifying cell viability, oxidative stress, ATP levels, mitochondrial membrane potential (MMP), mitochondrial calcium uptake, and autophagy.Results: DS and DSAD LCLs showed slower growth rates under minimal feeding. DS LCLs mainly dependent on mitochondrial respiration exhibited significantly slower growth and higher levels of oxidative stress compared to other groups. While ATP levels (under mitochondrial inhibitors) and mitochondrial calcium uptake were significantly reduced in DSAD and AD cells, MMP was decreased in DS, DSAD, and AD LCLs. Finally, DS LCLs showed markedly reduced levels of the autophagy marker LC3-II, underscoring the close association between metabolic dysfunction and impaired autophagy in DS.Conclusion: There are significant mitochondrial functional changes in LCLs derived from DS, DSAD, and AD patients. Several parameters analyzed were consistently different between DS, DSAD, and AD lines suggesting that metabolic indicators between LCL groups may be utilized as biomarkers of disease progression and/or treatment outcomes. [ABSTRACT FROM AUTHOR]

Amyloid-beta peptide accumulation in the brain is one of the main hallmarks of Alzheimer's disease. The amyloid aggregation process is associated with the generation of free radical species responsible for mitochondrial impairment and DNA damage that in turn activates poly(ADP-ribose)polymerase 1 (PARP-1). PARP-1 catalyzes the poly(ADP-ribosylation), a post-translational modification of proteins, cleaving the substrate NAD+ and transferring the ADP-ribose moieties to the enzyme itself or to an acceptor protein to form branched polymers of ADP-ribose. In this paper, we demonstrate that a mitochondrial dysfunction occurs in Alzheimer's transgenic mice TgCRND8, in SH-SY5Y treated with amyloid-beta and in 7PA2 cells. Moreover, PARP-1 activation contributes to the functional energetic decline affecting cytochrome oxidase IV protein levels, oxygen consumption rates, and membrane potential, resulting in cellular bioenergetic deficit. We also observed, for the first time, an increase of pyruvate kinase 2 expression, suggesting a modulation of the glycolytic pathway by PARP-1. PARP-1 inhibitors are able to restore both mitochondrial impairment and pyruvate kinase 2 expression. The overall data here presented indicate a pivotal role for this enzyme in the bioenergetic network of neuronal cells and open new perspectives for investigating molecular mechanisms underlying energy charge decline in Alzheimer's disease. In this scenario, PARP-1 inhibitors might represent a novel therapeutic intervention to rescue cellular energetic metabolism. [ABSTRACT FROM AUTHOR]

New Findings What is the central question of this study? Evidence from cellular and animal models suggests that SIRT3 is involved in regulating aerobic ATP production. Thus, we investigated whether changes in fatty acid and oxidative metabolism known to accompany fasting and exercise occur in association with changes in SIRT3 mitochondrial localization and expression in human skeletal muscle., What is the main finding and its importance? We find that 48 h of fasting and acute endurance exercise decrease SIRT3 mRNA expression but do not alter SIRT3 mitochondrial localization despite marked increases in fatty acid oxidation. This suggests that SIRT3 activity is not regulated by changes in mitochondrial localization in response to cellular energy stress in human skeletal muscle., The present study examined SIRT3 expression and SIRT3 mitochondrial localization in response to acute exercise and short-term fasting in human skeletal muscle. Experiment 1 involved eight healthy men (age, 21.4 ± 2.8 years; peak O2 uptake, 47.1 ± 11.8 ml min−1 kg−1) who performed a single bout of exercise at ∼55% of peak aerobic work rate for 1 h. Muscle biopsies were obtained at rest (Rest), immediately after exercise (EX-0) and 3 h postexercise (EX-3). Experiment 2 involved 10 healthy men (age, 22.0 ± 1.5 years; peak O2 uptake, 47.2 ± 6.7 ml min−1 kg−1) who underwent a 48 h fast, with muscle biopsies collected 1 h postprandial (Fed) and after 48 h of fasting (Fast). Mitochondrial respiration was measured using high-resolution respirometry in permeabilized muscle fibre bundles to assess substrate oxidation. Whole body fat oxidation increased after both exercise (Rest, 0.96 ± 0.32 kcal min−1; Exercise, 5.66 ± 1.97 kcal min−1; P < 0.001) and fasting (Fed, 0.87 ± 0.51 kcal min−1; Fast, 1.30 ± 0.37 kcal min−1, P < 0.05). SIRT3 gene expression decreased ( P < 0.05) after both exercise (−8%) and fasting (−19%); however, SIRT3 whole muscle protein content was unaltered after fasting. No changes were observed in SIRT3 mitochondrial localization following either exercise or fasting. Fasting also decreased the Vmax of glutamate [80 ± 43 versus 50 ± 21 pmol s−1 (mg dry weight)−1; P < 0.05]. These findings suggest that SIRT3 does not appear to be regulated by changes in mitochondrial localization at the time points measured in the present study in response to cellular energy stress in human skeletal muscle. [ABSTRACT FROM AUTHOR]

Background: Exacerbations of asthma caused by respiratory viral infections are serious conditions in need of novel treatment. To this end animal models of asthma exacerbations are warranted. We have shown that dsRNA challenges or rhinoviral infection produce exacerbation effects in mice with ovalbumin (OVA)-induced allergic asthma. However, house dust mite (HDM) is a more human asthma-relevant allergen than OVA. We thus hypothesised that dsRNA challenges in mice with HDM-induced experimental asthma would produce important translational features of asthma exacerbations.Method: Mouse airways were challenged locally with HDM or saline three times a week for three weeks to establish experimental asthma. Then daily local dsRNA challenges were given for three consecutive days to induce exacerbation. Bronchoalveolar lavage fluid (BALF) was analysed for inflammatory cells, total protein, the necrosis marker LDH and the alarmin ATP. Lung homogenates were analysed for mRNA expression (RT-qPCR) of TNF-α, CCL2, CCL5, IL-1β, IL-33, thymic stromal lymphopoietin (TSLP), and IL-25 as well as pattern recognition receptors (PRRs) RIG-I, MDA5 and TLR3. Lung tissue IL-33 was analysed with ELISA and PRRs were quantified by western blot. Immunohistochemistry indicated lung distribution of IL-33.Results: HDM challenge alone caused sustained increase in BALF total protein, eosinophils, lymphocytes and neutrophils, and transient increase in lung tissue expression of TSLP, IL-33 and TNF-α. dsRNA-induced exacerbation markedly and dose-dependently exaggerated these effects. Further, BALF levels of LDH and ATP, and lung tissue expression of CCL2, CCL5, IL-1β, IL-25 and PRRs were increased exclusively at the exacerbations. Lung protein levels of IL-33 were transiently increased by HDM and further increased at exacerbation.Conclusion: We demonstrate several novel aspects of HDM-induced experimental asthma and added exacerbation effects of dsRNA. General inflammatory parameters in BALF such as exuded proteins, mixed granulocytes, LDH and ATP were increased at the present exacerbations as they are in human asthma exacerbations. We suggest that this model of asthma exacerbation involving dsRNA challenges given to mice with established HDM-induced asthma has translational value and suggest that it may be particularly suited for in vivo studies involving pharmacological effects on exacerbation-induced expression of major upstream TH2-cytokines; IL-33, TSLP and IL-25, as well as PRRs. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Ibogaine is a naturally occurring alkaloid with psychotropic and metabotropic effects, derived from the bark of the root of the West African Tabernanthe iboga plant. The tribes of Kongo basin have been using iboga as a stimulant, for medicinal purposes, and in rite of passage ceremonies, for centuries. Besides, it has been found that this drug has anti-addictive effects. Aim of the study Previous studies have demonstrated that ibogaine changed the quantity of ATP and energy related enzymes as well as the activity of antioxidant enzymes in cells thus altering redox equilibrium in a time manner. In this work, the mechanism of its action was further studied by measuring the effects of ibogaine in human erythrocytes in vitro on ATP liberation, membrane fluidity and antioxidant enzymes activity. Materials and methods Heparinized human blood samples were incubated with ibogaine (10 and 20 μM) at 37°C for 1 h. Blood plasma was separated by centrifugation and the levels of ATP and uric acid were measured 10 min after the addition of ibogaine using standard kits. The activity of copper–zinc superoxide dismutase (SOD1), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR) were measured in erythrocytes after incubation period. The stability of SOD1 activity was further tested through in vitro incubation with H 2 O 2 and scanning of its electrophoretic profiles. Membrane fluidity was determined using an electron paramagnetic resonance spin-labelling method. Results Results showed that ibogaine treatment of erythrocytes in vitro increased ATP concentration in the blood plasma without changes in neither erythrocytes membrane fluidity nor uric acid concentration. Ibogaine also increased SOD1 activity in erythrocytes at both doses applied here. Treatment with 20 μM also elevated GR activity after in vitro incubation at 37 °C. Electrophoretic profiles revealed that incubation with ibogaine mitigates H 2 O 2 mediated suppression of SOD1 activity. Conclusion Some of the effects of ibogaine seem to be mediated through its influence on energy metabolism, redox active processes and the effects of discrete fluctuations of individual reactive oxygen species on different levels of enzyme activities. Overall, ibogaine acts as a pro-antioxidant by increasing activity of antioxidative enzymes and as an adaptagene in oxidative distress. [ABSTRACT FROM AUTHOR]

Background and Purpose: Although it is well accepted that cannabinoids modulate intestinal motility by reducing cholinergic neurotransmission mediated by CB(1) receptors, it is not known whether the endocannabinoids are involved in more complex circuits and if they interact with other systems. The aim of the present study was to examine possible interactions between cannabinoid CB(1) receptors and purines in the control of spontaneous contractility of longitudinal muscle in mouse ileum.Experimental Approach: The mechanical activity of longitudinally oriented ileal segments from mice was recorded as isometric contractions.Key Results: The selective CB(1) receptor agonist, N-(2-chloroethyl)5,8,11,14-eicosaetraenamide (ACEA) reduced, concentration dependently, spontaneous contractions in mouse ileum. This effect was almost abolished by tetrodotoxin (TTX) or atropine. Inhibition by ACEA was not affected by theophylline (P1 receptor antagonist) or by P2Y receptor desensitization with adenosine 5'[beta-thio]diphosphate trilithium salt, but was significantly reversed by pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) (P2 receptor antagonist), by P2X receptor desensitization with alpha,beta-methyleneadenosine 5'-triphosphate lithium salt (alpha,beta-MeATP) or by 8,8'-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino) bis(1,3,5-naphthalenetrisulphonic acid)] (P2X receptor antagonist). Contractile responses to alpha,beta-MeATP (P2X receptor agonist) were virtually abolished by TTX or atropine, suggesting that they were mediated by acetylcholine released from neurones, and significantly reduced by ACEA.Conclusion and Implications: In mouse ileum, activation of CB(1) receptors, apart from reducing acetylcholine release from cholinergic nerves, was able to modulate negatively, endogenous purinergic effects, mediated by P2X receptors, on cholinergic neurons. Our study provides evidence for a role of cannabinoids in the modulation of interneuronal purinergic transmission. [ABSTRACT FROM AUTHOR]

Investigations of training effects on exercise energy cost have yielded conflicting results. The purpose of the present study was to compare quadriceps energy cost and oxidative capacity between endurance-trained and sedentary subjects during a heavy dynamic knee extension exercise. We quantified the rates of ATP turnover from oxidative and anaerobic pathways with (31)P-MRS, and we measured simultaneously pulmonary oxygen uptake in order to assess both total ATP production [i.e., energy cost (EC)] and O(2) consumption (O(2) cost) scaled to power output. Seven sedentary (SED) and seven endurance-trained (TRA) subjects performed a dynamic standardized rest-exercise-recovery protocol at an exercise intensity corresponding to 35% of maximal voluntary contraction. We showed that during a dynamic heavy exercise, the O(2) cost and EC were similar in the SED and endurance-trained groups. For a given EC, endurance-trained subjects exhibited a higher relative mitochondrial contribution to ATP production at the muscle level (84 +/- 12% in TRA and 57 +/- 12% in SED; P < 0.01) whereas the anaerobic contribution was reduced (18 +/- 12% in TRA and 44 +/- 11% in SED; P < 0.01). Our results obtained in vivo illustrate that on the one hand the beneficial effects of endurance training are not related to any reduction in EC or O(2) cost and on the other hand that this similar EC was linked to a change regarding the contribution of anaerobic and oxidative processes to energy production, i.e., a greater aerobic energy contribution associated with a concomitant reduction of the anaerobic energy supply. [ABSTRACT FROM AUTHOR]

DNA topoisomerases are enzymes that disentangle the topological problems that arise in double-stranded DNA. Many of these can be solved by the generation of either single or double strand breaks. However, where there is a clear requirement to alter DNA topology by introducing transient double strand breaks, only DNA topoisomerase II (TOP2) can carry out this reaction. Extensive biochemical and structural studies have provided detailed models of how TOP2 alters DNA structure, and recent molecular studies have greatly expanded knowledge of the biological contexts in which TOP2 functions, such as DNA replication, transcription and chromosome segregation -- processes that are essential for preventing tumorigenesis. [ABSTRACT FROM AUTHOR]

Abstract: Nucleotide signaling is currently an area of intense investigation. Extracellular adenosine triphosphate (ATP) liberated during hypoxia or inflammation can either signal directly to purinergic receptors or, after phosphohydrolytic metabolism, can activate surface adenosine receptors. Given the association of polymorphonuclear leukocytes (PMNs) with adenine nucleotide/nucleoside signaling in the inflammatory milieu, it was recently demonstrated that PMNs actively release ATP via a connexin 43 hemichannel-dependent mechanism. Here, we review the mechanisms of ATP release and subsequent functional implications of ATP metabolism at the interface between PMN and vascular endothelial cells during inflammation and in hypoxia. [Copyright &y& Elsevier]

Recent studies have suggested a role for a chloride current in the modulation of pacemaker potentials generated by interstitial cells of Cajal. Patch-clamp recordings were made from inside-out patches of cultured interstitial cells of Cajal from the murine small intestine. The majority of patches were quiescent immediately after excision, but in some patches currents activated spontaneously after a period of 10 min to 1 h. Currents could also be activated by strongly polarizing the patch. It was found that the currents activated in both cases included a chloride channel. This channel could also be activated by ATP and the catalytic subunit of protein kinase A. The channel had conductance states (+/-SD) of 53 +/- 25.35, 126 +/- 21.44, 180 +/- 12.57 and 211 +/- 8.86 pS. It was outwardly rectifying (as a function of open probability) and deactivated (i.e., gave a tail current) but showed no inactivation. The permeability sequence of the channel was I(-)>>Br(-)>or=Cl(-)>Asp(-). It was unaffected in magnitude or rectification by changing the free Ca2+ concentration of the bath between <10 nM, 100 nM (control) and 2 mM . [ABSTRACT FROM AUTHOR]

Objective: Chondrocytes are highly sensitive to variations in extracellular glucose and oxygen levels in the extracellular matrix. As such, they must possess a number of mechanisms to detect and respond to alterations in the metabolic state of cartilage. In other organs such as the pancreas, heart and brain, such detection is partly mediated by a family of potassium channels known as K(ATP) (adenosine 5'-triphosphate-sensitive potassium) channels. Here we investigate whether chondrocytes too express functional K(ATP) channels, which might, potentially, serve to couple metabolic state with cell activity.Methods: Immunohistochemistry was used to explore K(ATP) channel expression in equine and human chondrocytes. Biophysical properties of equine chondrocyte K(ATP) channels were investigated with patch-clamp electrophysiology.Results: Polyclonal antibodies directed against the K(ATP) Kir6.1 subunit revealed high levels of expression in human and equine chondrocytes mainly in superficial and middle zones of normal cartilage. Kir6.1 was also detected in superficial chondrocytes in osteoarthritic (OA) cartilage. In single-channel electrophysiological studies of equine chondrocytes, we found K(ATP) channels to have a maximum unitary conductance of 47 +/- 9 pS (n=5) and a density of expression comparable to that seen in excitable cells.Conclusion: We have shown, for the first time, functional K(ATP) channels in chondrocytes. This suggests that K(ATP) channels are involved in coupling metabolic and electrical activities in chondrocytes through sensing of extracellular glucose and intracellular adenosine triphosphate (ATP) levels. Altered K(ATP) channel expression in OA chondrocytes may result in impaired intracellular ATP sensing and optimal metabolic regulation. [ABSTRACT FROM AUTHOR]

Summary: Objective: Pro-inflammatory cytokines play an important role in osteoarthritis (OA). In osteoarthritic cartilage, chondrocytes exhibit an alteration in mitochondrial activity. This study analyzes the effect of tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) on the mitochondrial activity of normal human chondrocytes. Materials and methods: Mitochondrial function was evaluated by analyzing the activities of respiratory chain enzyme complexes and citrate synthase, as well as by mitochondrial membrane potential (Δψ m) and adenosine triphosphate (ATP) synthesis. Bcl-2 family mRNA expression and protein synthesis were analyzed by RNase protection assay (RPA) and Western-blot, respectively. Cell viability was analyzed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and apoptosis by 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) stain. Glycosaminoglycans were quantified in supernatant by a dimethyl-methylene blue binding assay. Results: Compared to basal cells, stimulation with TNFα (10ng/ml) and IL-1β (5ng/ml) for 48h significantly decreased the activity of complex I (TNFα=35% and IL-1β=35%) and the production of ATP (TNFα=18% and IL-1β=19%). Both TNFα and IL-1β caused a definitive time-dependent decrease in the red/green fluorescence ratio in chondrocytes, indicating depolarization of the mitochondria. Both cytokines induced mRNA expression and protein synthesis of the Bcl-2 family. Rotenone, an inhibitor of complex I, caused a significant reduction of the red/green ratio, but it did not reduce the viability of the chondrocytes. Rotenone also increased Bcl-2 mRNA expression and protein synthesis. Finally, rotenone as well as TNFα and IL-1β, reduced the content of proteoglycans in the extracellular matrix of normal cartilage. Conclusion: These results show that both TNFα and IL-1β regulate mitochondrial function in human articular chondrocytes. Furthermore, the inhibition of complex I by both cytokines could play a key role in cartilage degradation induced by TNFα and IL-1β. These data could be important for understanding of the OA pathogenesis. [Copyright &y& Elsevier]

K(+) channels play an important role in pump-leak coupling and volume regulation in the renal proximal tubule. Previous experiments have identified a barium-sensitive K(+) conductance (G(Ba)) in proximal tubule cells isolated from frog kidneys. In this paper we examine the regulation of G(Ba) by ATP. G(Ba) was measured in single cells isolated from frog kidney using the whole-cell patch-clamp technique. G(Ba) was activated by 2 mM: intracellular ATP. This activation was enhanced by inhibition of protein kinase C and attenuated by inhibition of protein kinase A, indicating reciprocal regulation by these kinases. Activation by ATP was reduced in the presence of a hypertonic bath solution, suggesting that cell swelling was required. However, after activation to steady-state, G(Ba )was not sensitive to cell-volume changes. Hypotonic shock-induced volume regulation was inhibited by barium and quinidine, inhibitors of G(Ba). The effect of maximal inhibitory concentrations of barium and quinidine on volume regulation was similar and addition of both blockers together did not augment the inhibitory response. G(Ba) was also activated by ADP, via a mechanism dependent on the presence of Mg(2+). However, the responses to ADP and ATP were not additive, suggesting that these nucleotides may share a common mechanism of activation. The regulation of G(Ba) by ATP was biphasic, with a half-maximal activating concentration of 0.89 mM and a half maximal inhibitory concentration of 6.71 mM. The sensitivity to nucleotides suggests that G(Ba) may be regulated by the metabolic state of the cell. Furthermore, the sensitivity to solution osmolality, coupled with the blocker profile of inhibition of volume regulation, suggests that G(Ba) could play a role in volume regulation. [ABSTRACT FROM AUTHOR]

The ATP-sensitive K(+) (K(ATP)) channels couple chemical signals to cellular activity, in which the control of channel opening and closure (i.e., channel gating) is crucial. Transmembrane helices play an important role in channel gating. Here we report that the gating of Kir6.2, the core subunit of pancreatic and cardiac K(ATP) channels, can be switched by manipulating the interaction between two residues located in transmembrane domains (TM) 1 and 2 of the channel protein. The Kir6.2 channel is gated by ATP and proton, which inhibit and activate the channel, respectively. The channel gating involves two residues, namely, Thr71 and Cys166, located at the interface of the TM1 and TM2. Creation of electrostatic attraction between these sites reverses the channel gating, which makes the ATP an activator and proton an inhibitor of the channel. Electrostatic repulsion with two acidic residues retains or even enhances the wild-type channel gating. A similar switch of the pH-dependent channel gating was observed in the Kir2.1 channel, which is normally pH- insensitive. Thus, the manner in which the TM1 and TM2 helices interact appears to determine whether the channels are open or closed following ligand binding. [ABSTRACT FROM AUTHOR]

Although nuclear pore complexes (NPC) are considered to be key structures in gene expression, little is known about their regulatory control. In order to explore the regulatory mechanism of passive transport of small macromolecules we examined the influence of different factors on the diffusional pathway of NPCs in isolated Xenopus laevis oocyte nuclei. Diffusion of fluorescence-labeled 10-kD dextran was measured across the nuclear envelope with confocal fluorescence microscopy. Surprisingly, the filling state of the perinuclear Ca(2+) store had no influence on passive transport of 10-kD dextran. Furthermore, nuclear envelope permeability was independent of cytoplasmic pH (pH range 8.3-6.3). In contrast, nuclear swelling, induced by omission of the endogenous cytosolic macromolecules, clearly increased nuclear permeability. An antibody against the glycoprotein gp62, located at the central channel entrance, reduced macromolecule diffusion. In addition, nuclei from transcriptionally active, early developmental stages (stage II) were less permeable compared to transcriptionally inactive, late-developmental-stage (stage VI) nuclei. In stage II nuclei, atomic force microscopy disclosed NPC central channels with plugs that most likely were ribonucleoproteins exiting the nucleus. In conclusion, the difference between macromolecule permeability and previous measurements of electrical resistance strongly indicates separate routes for macromolecules and ions across the nuclear envelope. [ABSTRACT FROM AUTHOR]

Background: The relative magnitude of the inner mitochondrial membrane potential (DeltaPsim) has been suggested to indicate the competence of mammalian gametes and early embryos. This study examined the response of cultured somatic cells and mouse oocytes to inhibitors and conditions that affect DeltaPsim or metabolism, or both, and measured treatment-specific changes in ATP and cytoplasmic free Ca(2+).Methods: During and after treatment, relative DeltaPsim, free Ca(2+), and ATP levels and cortical granule density were determined.Results: Comparable responses of somatic cells and metaphase II mouse oocytes to experimental manipulations that affect DeltaPsim and metabolism were observed and reversible loss of DeltaPsim was associated with increased intracellular free Ca(2+), which in certain instances resulted in parthenogenetic activation.Conclusion: The findings support a mitochondrial basis for pericortical J-aggregate fluorescence but not for a direct association between high DeltaPsim and metabolism. The results extend previous findings indicating that high-polarized (high DeltaPsim, JC-1 J-aggregate-forming) mitochondria occur in pericortical domains in mouse and human oocytes and early preimplantation stage embryos and support the notion that this spatial distribution may be related to localized ionic and metabolic regulation. [ABSTRACT FROM AUTHOR]

Background: Sulfonylurea agents exert their physiological effects in many cell types via binding to specific sulfonylurea receptors (SUR). SUR couple to inwardly-rectifying K+ channel (Kir6.x) to form tetradimeric ATP-sensitive K+ channels (KATP). The SUR subunits confer ATP-sensitivity on KATP and also provide the binding sites for sulfonylureas and other pharmacological agents. Our previous work demonstrated that the exposure of mesangial cells (MC) to sulfonylureas generated profound effects on MC glucose uptake and matrix metabolism and induced heightened cell contractility in association with Ca2+ transients. Because these responses likely resulted from the binding of sulfonylurea to a mesangial SUR2, we subsequently documented [3H]-glibenclamide binding to MC and the gene expression of several mesangial SUR2 transcripts. From these data, we inferred that MC expressed the components of a mesangial KATP and sought to establish their presence in primary MC.Methods: To obtain mesangial SUR2 cDNA sequences, rapid amplification of cDNA ends (RACE) was utilized. DNA sequences were established by the fluorescent dye termination method. Gene expression of mesangial SUR2 and Kir6.1/2 was examined by reverse transcription polymerase chain reaction (RT-PCR) and Northern analysis. SUR2 proteins were identified by immunoblotting of mesangial proteins from membrane-enriched fractions with polyclonal antiserum directed against SUR2.Results: RACE cloning yielded two mesangial SUR2 cDNAs of 4.8 and 6.7 kbp whose open reading frames translated proteins of 964 and 1535 aa, respectively. Using probes specific to each cDNA, the presence of a unique, 5.5 kbp serum-regulated mesangial SUR2 splice variant was established. The sequence of this mesangial SUR2 (mcSUR2B) shares identity with the recently cloned rat SUR2B (rSUR2B), but, in comparison to rSUR2B, is truncated by 12 exons at the N-terminus where it contains a unique insert of 16 aa. Immunoblotting studies with anti-SUR2 antiserum demonstrated SUR2 proteins of 108 and 170 kD in membrane-enriched fractions of MC protein extracts. Complementary studies showed abundant gene expression of Kir6.1, thereby establishing gene expression of both components of KATP.Conclusions: Based upon analogy to vascular smooth muscle cells (VSMC), there are at least two putative mesangial KATP that most likely represent hetero-octamers, comprised of either rSUR2B or mcSUR2 in complex with Kir6.1. Our results define the mesangial SUR2B as the possible first link in a chain of cellular events that culminates in MC contraction and altered extracellular matrix metabolism following exposure to sulfonylureas. In addition, our results serve as the basis for the future elucidation of the electrophysiologic characteristics of the mesangial KATP and the study of endogenous regulators of mesangial cell contractility. [ABSTRACT FROM AUTHOR]

The insulin response to the sulfonylurea glibenclamide was markedly impaired in pancreatic beta-cell line MIN6 cells with chronic glibenclamide treatment (MIN6-Glib). The intracellular calcium concentration increased only slightly in response to glibenclamide in MIN6-Glib. While the properties of the voltage-dependent calcium channels were not altered, the conductance of the K(ATP) channels, the primary target of glibenclamide, was significantly reduced in MIN6-Glib. The ATP-sensitive K+ (K(ATP)) channels in MIN6 cells comprise inwardly rectifying K+ channel member Kir6.2 subunits and sulfonylurea receptor (SUR) 1 subunits. MIN6 cells have both high- and low-affinity binding sites for glibenclamide. The binding affinities at these two sites were unchanged, but the maximum binding capacities at both sites were similarly increased by chronic glibenclamide treatment. Both SUR1 and Kir6.2 mRNA levels were not altered, but SUR1 protein was rather increased in MIN6-Glib. In addition, electron microscopic examination revealed a majority of the SUR1 to be present in a cluster near the plasma membrane in control MIN6, while it tends to be distributed in the cytoplasm in MIN6-Glib. These data suggest that chronic glibenclamide treatment causes the defect in acute glibenclamide-induced insulin secretion by reducing the number of functional K(ATP) channels on the plasma membrane of the beta-cells. [ABSTRACT FROM AUTHOR]

The digestibility of D-tagatose, its effect on the digestibility of macronutrients and the metabolic response of the microbiota of the gastrointestinal tract to the ingestion of this carbohydrate were studied in pigs. Eight pigs were fed a low fiber diet comprising 15% sucrose (control group). Another eight pigs were fed a similar diet except that 100 g sucrose per kg diet was replaced by D-tagatose (test group). After 18 d, the pigs were killed and the gastrointestinal contents removed for analysis. The digestibility of D-tagatose was 25.8 +/- 5.6% in the distal third of the small intestine. The small intestinal digestibilities of dry matter (86.9 +/- 1.3 vs. 92.9 +/- 0.9%), gross energy (74.4 +/- 1.6 vs. 80.7 +/- 1.8%) and sucrose (90.4 +/- 2.5 vs. 98.0 +/- 0.5%) were lower (P < 0. 05) in the pigs fed D-tagatose. Digestibilities of starch, protein and fat did not differ between groups. D-Tagatose, sucrose and starch were fully digested in the large intestine. The fecal digestibilities of energy, dry matter and fat did not differ between the two groups, whereas D-tagatose reduced the fecal digestibility of protein (91.1 +/- 0.6 vs. 93.5 +/- 0.7%, P < 0.05). D-Tagatose served as a substrate for the microbiota in the cecum and proximal colon as indicated by a reduced pH, and a greater ATP concentration, adenylate energy charge (AEC) ratio and concentration of short-chain fatty acids. In particular, the increase in the concentrations of propionate, butyrate and valerate suggests possible health benefits of this monosaccharide. [ABSTRACT FROM AUTHOR]

(H,K)-ATPase containing membranes from hog stomach were attached to black lipid membranes. Currents induced by an ATP concentration jump were recorded and analyzed. A sum of three exponentials (tau 1(-1) approximately 400 sec-1, tau 2(-1) approximately 100 sec-1, tau 3(-1) approximately 10 sec-1; T = 300 K, pH 6, MgCl2 3 mM, no K+) was fitted to the transient signal. The dependence of the resulting time constants and the peak current on electrolyte composition, ATP conversion rate, temperature, and membrane conductivity was recorded. The results are consistent with a reaction scheme similar to that proposed by Albers and Post for the NaK-ATPase. Based on this model the following assignments were made: tau 2 corresponds to ATP binding and exchange with caged ATP. tau 1 describes the phosphorylation reaction E1 x ATP-->E1P. The third, slowest time constant tau 3 is tentatively assigned to the E1P-->E2P transition. This is the first electrogenic step and is accelerated at high pH and by ATP via a low affinity binding site. The second electrogenic step is the transition from E2K to E1H. The E2K<==>E1H equilibrium is influenced by potassium with an apparent K0.5 of 3 mM and by the pH. Low pH and low potassium concentration stabilize the E1 conformation. [ABSTRACT FROM AUTHOR]

The effects of tetraethylammonium (TEA) and quinine on Ca-activated [K(Ca)], ATP-sensitive [K(ATP)]K channels and delayed-rectifier K current [K(dr)] have been studied in cultured insulin-secreting HIT cells using the patch-clamp technique. K(Ca) and K(ATP) channels were identified in excised, outside/out patches using physiological solutions and had unitary conductances of 60.8 +/- 1.3 pS (n = 31) and 15.4 +/- 0.3 pS (n = 40), respectively. Macroscopic K(dr) current (peak current = 607 +/- 100 pA at +50 mV, n = 14) were recorded in the presence of 100 microM cadmium and 0.5 microM tetrodotoxin. Tetraethylammonium (TEA) blocked all three channel types but was more effective on K(Ca) channels (EC50 = 0.15 mM) than on K(ATP) channels (EC50 = 15 mM) or K(dr) currents (EC50 = 3 mM). Quinine also blocked all three currents but was less effective on K(Ca) channels (EC50 = 0.3 mM) while equally effective against K(ATP) channels and K(dr) currents (EC50 = 0.025 mM). TEA blocked K(Ca) and K(ATP) channels by reducing their single-channel conductances and decreasing the probability of K(ATP) channel opening. Quinine blocked K(Ca) channels by reducing the single-channel conductance, but blocked K(ATP) channels by reducing the probability of channel opening. Reinterpretation of previous microelectrode studies in light of these findings suggest that, (i) only K(ATP) channels are active in low glucose, (ii) both K(Ca) and K(dr) channels may assist Ca-spike repolarization, and (iii) K(Ca) channels play no role in forming the burst pattern of Ca spiking in the B cell. [ABSTRACT FROM AUTHOR]

It is generally agreed that solute transport across the Chara plasma membrane is energized by a proton electrochemical gradient maintained by an H(+)-extruding ATPase. Nonetheless, as deduced from steady-state current-voltage (I-V) measurements, the kinetic and thermodynamic constraints on H(+)-ATPase function remain in dispute. Uncertainties necessarily surround long-term effects of the relatively nonspecific antagonists used in the past; but a second, and potentially more serious problem has sprung from the custom of subtracting, across the voltage spectrum, currents recorded following pump inhibition from currents measured in the control. This practice must fail to yield the true I-V profile for the pump when treatments alter the thermodynamic pressure on transport. We have reviewed these issues, using rapid metabolic blockade with cyanide and fitting the resultant whole-cell I-V and difference-current-voltage (dI-V) relations to a reaction kinetic model for the pump and parallel, ensemble leak. Measurements were carried out after blocking excitation with LaCl3, so that steady-state currents could be recorded under voltage clamp between -400 and +100 mV. Exposures to 1 mM NaCN (CN) and 0.4 mM salicylhydroxamic acid (SHAM) depolarized (positive-going) Chara membrane potentials by 44-112 mV with a mean half time of 5.4 +/- 0.8 sec (n = 13). ATP contents, which were followed in parallel experiments, decayed coincidently with a mean half time of 5.3 +/- 0.9 sec [( ATP]t = 0, 0.74 +/- 0.3 mM; [ATP]t = infinity, 0.23 +/- 0.02 mM). Current-voltage response to metabolic blockade was described quantitatively in context of these changes in ATP content and the consequent reduction in pump turnover rate accompanied by variable declines in ensemble leak conductance. Analyses of dI-V curves (+/- CN + SHAM) as well as of families of I-V curves taken at times during CN + SHAM exposures indicated a stoichiometry for the pump of one charge (H+) transported per ATP hydrolyzed and an equilibrium potential near -420 mV at neutral external pH; under these conditions, the pump accounted for approximately 60-75% of the total membrane conductance near Vm. Complementary results were obtained also in fitting previously published I-V data gathered over the external pH range 4.5-7.5. Kinetic features deduced for the pump were dominated by a slow step preceding H+ unloading outside, and by recycling and loading steps on the inside which were in rapid equilibrium.(ABSTRACT TRUNCATED AT 400 WORDS) [ABSTRACT FROM AUTHOR]

Nonstationary electric currents are described which are generated by the Na,K-pump. Flat membrane sheets 0.2-1 micron in diameter containing a high density of oriented Na,K-ATPase molecules are bound to a planar lipid bilayer acting as a capacitive electrode. In the aqueous phase adjacent to the bound membrane sheets, ATP is released within milliseconds from an inactive, photolabile precursor ("caged" ATP) by an intense flash of light. After the ATP-concentration jump, transient current and voltage signals can be recorded in the external circuit corresponding to a translocation of positive charge across the pump protein from the cytoplasmic to the extracellular side. These electrical signals which can be suppressed by inhibitors of the Na,K-ATPase require the presence of Na+ but not of K+ in the aqueous medium. The intrinsic pump current Ip(t) can be evaluated from the recorded current signal, using estimated values of the circuit parameters of the compound membrane system. Ip(t) exhibits a biphasic behavior with a fast rising period, followed by a slower decline towards a small quasi-stationary current. The time constant of the rising phase of Ip(t) is found to depend on the rate of photochemical ATP release. Further information on the microscopic origin of the current transient can be obtained by double-flash experiments and by chymotrypsin modification of the protein. These and other experiments indicate that the observed charge-translocation is associated with early events in the normal transport cycle. After activation by ATP, the pump goes through the first steps of the cycle and then enters a long-lived state from which return to the initial state is slow. [ABSTRACT FROM AUTHOR]

The use of an Ussing chamber with well-defined mixing characteristics coupled to a mass spectrometer permits the concurrent evaluation of transepithelial current and oxidative metabolism with improved temporal resolution. The time-course of the amiloride-sensitive current Ia and the rate of suprabasal CO2 production JsbCO2 were observed in 10 toad urinary bladders at short-circuit and after clamping delta psi at 100 mV, serosa positive. Following perturbation of delta psi (0 leads to 100 mV), Ia declined sharply within 1/2 min, remaining near constant approximately 15 min, and then increased slightly. JsbCO2 declined more gradually, remained near constant at approximately 4-7 min, and then declined further. Detailed analysis revealed an early quasi-steady state with near constancy of JsbCO2 starting at 2.9 +/- 1.1 (SD) min and lasting 4.7 +/- 1.8 (SD) min, followed by relaxation to a later steady state at about 15 min. During the early quasi-steady state, Ia was also nearly constant. Considering that in steady states Ia/F approximately or equal to JaNa, the rate of transepithelial active Na transport, during the early quasi-steady state mean values +/- SE of JaNa, JsbCO2 and (JaNa/JsbCO2) were, respectively, 29.9 +/- 1.7%, 59.4 +/- 3.2%, and 56.4 +/- 5.7% of values at short-circuit. Corresponding values during the late steady state were 41.4 +/- 6.0%, 38.2 +/- 6.1%, and 111.3 +/- 8.6%. Thus the flow ratio JaNa/JsbCO2 was depressed significantly during the early quasi-steady state, but returned later to the original value. The results of measurements of Ia and JsbO2 in three hemibladders were qualitatively similar. In terms of a phenomenological "black-box" treatment the findings are consistent with earlier studies indicating incomplete coupling between transport and metabolism. Further studies will be required to clarify the molecular basis for these observations. [ABSTRACT FROM AUTHOR]