Sodium-potassium Pump Activity Research Articles

Resting membrane potential and intracellular [Na+] at rest, during fatigue and during recovery in rat soleus muscle fibres in situ.

Large trans-sarcolemmal ionic shifts occur with fatiguing exercise or stimulation of isolated muscles. However, it is unknown how resting membrane potential (EM) and intracellular sodium concentration ([Na+]i) change with repeated contractions in living mammals. We investigated (i) whether [Na+]i (peak, kinetics) can reveal changes of Na+-K+ pump activity during brief or fatiguing stimulation and (ii) how resting EM and [Na+]i change during fatigue and recovery of rat soleus muscle in situ. Muscles of anaesthetised rats were stimulated with brief (10s) or repeated tetani (60Hz for 200ms, every 2s, for 30s or 300s) with isometric force measured. Double-barrelled ion-sensitive microelectrodes were used to quantify resting EM and [Na+]i. Post-stimulation data were fitted using polynomials and back-extrapolated to time zero recovery. Mean pre-stimulation resting EM (layer 2-7 fibres) was -71mV (surface fibres were more depolarised), and [Na+]i was 14mM. With deeper fibres, 10s stimulation (2-150Hz) increased [Na+]i to 38-46mM whilst simultaneously causing hyperpolarisations (7.3mV for 2-90Hz). Fatiguing stimulation for 30s or 300s led to end-stimulation resting EM of -61 to -53mV, which recovered rapidly (T1/2, 8-22s). Mean end-stimulation [Na+]i increased to 86-101mM with both fatigue protocols and the [Na+]i recovery time-course (T1/2, 21-35s) showed no difference between protocols. These combined findings suggest that brief stimulation hyperpolarises the resting EM, likely via maximum Na+-induced stimulation of the Na+-K+ pump. Repeated tetani caused massive depolarisation and elevations of [Na+]i that together lower force, although they likely interact with other factors to cause fatigue. [Na+]i recovery kinetics provided no evidence of impaired Na+-K+ pump activity with fatigue. KEY POINTS: It is uncertain how resting membrane potential, intracellular sodium concentration ([Na+]i), and sodium-potassium (Na+-K+) pump activity change during repeated muscle contractions in living mammals. For rat soleus muscle fibres in situ, brief tetanic stimulation for 10s led to raised [Na+]i, anticipated to evoke maximal Na+-induced stimulation of the Na+-K+ pump causing an immediate hyperpolarisation of the sarcolemma. More prolonged stimulation with repeated tetanic contractions causes massive elevations of [Na+]i, which together with large depolarisations (via K+ disturbances) likely reduce force production. These effects occurred without impairment of Na+-K+ pump function. Together these findings suggest that rapid activation of the Na+-K+ pump occurs with brief stimulation to maintain excitability, whereas more prolonged stimulation causes rundown of the trans-sarcolemmal K+ gradient (hence depolarisation) and Na+ gradient, which in combination can impair contraction to contribute to fatigue in living mammals.

Branchial bioenergetics dysfunction as a relevant pathophysiological mechanism in freshwater silver catfish (Rhamdia quelen) experimentally infected with Flavobacterium columnare

Flavobacterium columnare, the causative agent of columnaris disease, is a serious bacterial disease responsible for causing devastating mortality rates in several species of freshwater fish, leading to severe economic losses in the aquaculture industry. Notwithstanding the enormous impacts this disease can have, very little is known regarding the interaction between the host and bacterium in terms of the mortality rate of silver catfish (Rhamdia quelen), as well its linkage to gill energetic homeostasis. Therefore, we conducted independent experiments to evaluate the mortality rates caused by F. columnare in silver catfish, as well as whether columnaris disease impairs the enzymes of the phosphoryl transfer network in gills of silver catfish and the pathways involved in this inhibition. Experiment I revealed that clinical signs started to appear 72 h post-infection (hpi), manifesting as lethargy, skin necrosis, fin erosion and gill discoloration. Silver catfish began to die at 96 hpi, and 100% mortality was observed at 120 hpi. Experiment II revealed that creatine kinase (CK, cytosolic and mitochondrial) and pyruvate kinase (PK) activities were inhibited in silver catfish experimentally infected with F. columnare, while no significant difference was observed between experimental and control groups with respect to adenylate kinase activity. Activity of the branchial sodium-potassium pump (Na+, K+-ATPase) was inhibited while reactive oxygen species (ROS) and lipid peroxidation levels were higher in silver catfish experimentally infected with F. columnare than in the control group at 72 hpi. Based on these data, the impairment of CK activity elicited by F. columnare caused a disruption in branchial energetic balance, possibly reducing ATP availability in the gills and provoking impairment of Na+, K +ATPase activity. The inhibition of CK and PK activities appears to be mediated by ROS overproduction and lipid peroxidation, both of which contribute to disease pathogenesis associated with branchial tissue.

Disturbance of energetic homeostasis and oxidative damage provoked by trichlorfon as relevant toxicological mechanisms using silver catfish as experimental model

Recent evidences have suggested the involvement of phosphoryl transfer, catalyzed by creatine kinase (CK), adenylate kinase (AK) and pyruvate kinase (PK), to metabolic alterations and impairment of bioenergetics homeostasis linked to adenosine triphosphate (ATP) production, and utilization during exposure to pesticides. It is recognized that sublethal concentrations of trichlorfon alter hepatic and branchial metabolism, but the pathways involved in this process remains unknown. Thus, the aim of this study was to evaluate whether phosphoryl transfer network can be a pathway involved in the hepatic and branchial metabolic alterations during exposure to sublethal concentrations of trichlorfon using silver catfish Rhamdia quelen as experimental model. Hepatic and branchial CK (cytosolic and mitochondrial isoforms) and PK activities were inhibited after 48 h of exposure to 11 and 22 mg/L trichlorfon compared to control group, while AK activity did not differ between groups. In addition, sodium-potassium pump (Na+, K+-ATPase) activity was lower after 48 h of exposure to 22 mg/L trichlorfon compared to control group. Thiobarbituric acid reactive substances (TBARS) were higher in liver samples after 24 h of exposure to 22 mg/L trichlorfon compared to control group, as well as after 48 h of exposure to 11 and 22 mg/L trichlorfon in liver and gills. Finally, hepatic and branchial non-protein thiol (NPSH) levels were lower after 48 h of exposure to 11 and 22 mg/L trichlorfon. All evaluated parameters did not recover after 48 h in clean water. Based on these evidence, the impairment of phosphoryl transfer network can be considered a pathway involved in the hepatic and branchial metabolic alterations during exposure to sublethal concentrations of trichlorfon. Moreover, alterations on CK and PK activities provoke an impairment on Na+, K+-ATPase activity, which can be mediated by lipid oxidative damage and reduction of NPSH content.

Blood-brain barrier breakdown, memory impairment and neurotoxicity caused in mice submitted to orally treatment with thymol

Several evidences have related the biochemical and pharmacological properties of thymol, but the possible neurotoxic effects of this compound remain unknown and not evaluated. Thus, the purpose of this study was to evaluate whether intake of thymol in different doses (10, 20 and 40 mg/kg) induce neurotoxicity and behavioral alterations using mice as experimental model, as well as the involvement of blood-brain barrier (BBB) and brain neurotransmitters in these alterations. Thymol (20 and 40 mg/kg) significantly decrease latency time to inhibitory avoidance task when compared to control group, indicating a memory loss after 30 days of oral treatment. Also, thymol (20 and 40 mg/kg) induced a significant increase on BBB permeability to Evan’s blue dye when compared to control group, which is an indicative of BBB breakdown. Moreover, a significant increase of brain acetylcholinesterase (AChE) was observed in mice treated with 40 mg/kg of thymol, while the activity of sodium-potassium pump (Na+, K+-ATPase) was inhibited in mice treated with 20 and 40 mg/kg thymol when compared to control group. Finally, mice that received 20 and 40 mg/kg thymol showed a significant increase on cerebral reactive oxygen species (ROS) levels and cerebral xanthine oxidase (XO) activity compared to control group. Based on these evidences, the rupture of BBB can be considered an important pathway linked in thymol-induced memory loss. Also, the augmentation of brain ROS levels elicited by increase on XO activity may be a via involved in the damage to BBB, and an oxidative pathway that impairs the activity of brain neurotransmitters, as AChE and Na+, K+-ATPase. In summary, the dose of 10 mg/kg thymol can be safe and without neurotoxic effects in a period of 30 days of intake.

Oxidative stress mediated the inhibition of cerebral creatine kinase activity in silver catfish fed with aflatoxin B1-contaminated diet.

Aflatoxin B1 (AFB1) is an environmental toxicant and neurotoxic compound that induces the production of free radicals, causing oxidative stress. Creatine kinase (CK) is a central controller of energy metabolism in tissues with a large and fluctuating energy demand, and it is highly susceptible to inactivation by free radicals and oxidative damage. Thus, the aim of this study was to evaluate whether a diet for freshwater silver catfish (Rhamdia quelen) containing AFB1 inhibits cerebral CK activity, as well as the involvement of the oxidative stress on this inhibition. Brain CK activity was lower on days 14 and 21 post-feeding in animals that received AFB1-contaminated diet compared to the control group (basal diet), similarly to the brain sodium-potassium pump (Na+, K+-ATPase) activity. On the other hand, lipid peroxidation and protein carbonylation levels were higher on days 14 and 21 post-feeding in animals fed with AFB1-contaminated feed compared to the control group, while the antioxidant capacity against peroxyl radicals and thiol content was lower. Based on these evidences, the data demonstrated that diet containing AFB1 severely affects CK activity, an essential enzyme that plays an important role in brain energy homeostasis. Also, the impairment of energetic homeostasis linked with the use and generation of ATP via inhibition of CK activity elicited an inhibition of enzymes ATP-dependent, such as Na+, K+-ATPase. Moreover, the inhibition of brain CK activity appears to be mediated by the oxidation of lipids, proteins, and thiol group, as well as by a reduction in the antioxidant capacity.

Ouabain-Induced Stimulation of Sodium Potassium Pump Activity is Transient in Cardiac Myocytes

Cardiotonic steroids (CTS) unequivocally inhibit the sodium-potassium pump (NKP), but reports exist of stimulation at low concentrations. The effect at low doses is critical for interpretation of the extensive literature on circulating endogenously synthesised CTS. We have examined the effects of CTS on NKP activity at low doses with emphasis on ouabain, which is widely reported to be synthesised endogenously. Using the whole-cell patch clamp technique we voltage-clamped rabbit cardiomyocytes. Experimental solutions blocked non-NKP membrane currents and pump current (Ip) was identified. Myocytes were exposed to CTS for ∼1 min before Ip was measured or first preincubated for ∼1 hour. Exposure to 1–30 nM ouabain for ∼1 min maximally increased Ip at 5 nM to 0.69 ± 0.09 (n = 6) versus 0.46 ± 0.03 pA/pF (n = 25) in controls (p ≤ 0.01). Co-exposure to 500 nM rostafuroxin, a putative ouabain antagonist, abolished this stimulation (Ip = 0.40 ± 0.04 pA/pF; n = 7). Ouabagenin, which lacks the sugar moiety of ouabain, did not significantly increase Ip between 10 and 500 nM, nor did dihydroouabain at 1–50 nM, or digoxin at 0–30 nM. Dihydroouabain differs from ouabain by having a saturated lactone ring, and digoxin shares only the lactone ring of ouabain. Steady-state binding of ouabain will not occur with the ∼1 min exposures, so we pre-exposed myocytes for 1 hour to achieve near steady-state binding. Ouabain significantly reduced Ip (p ≤ 0.05), even at concentrations ≤0.25 nM that are relevant to reported endogenous serum levels. While ouabain can stimulate the NKP, stimulation is transient. It may occur during a relatively slow transition of ouabain through its access pathway before it reaches the inhibitory site.

Abstract 90: Inimitable Ouabain: The Endogenous Circulating Cardiotonic Steroid that Singularly Stimulates Sodium Potassium Pump Activity at Low Doses

Rationale: Cardiotonic steroids (CTS), such as digoxin, have been used to treat heart failure (HF) for over 200 years. They inhibit the sodium-potassium pump (NKP), and increase cardiac contractility by inhibiting efflux of sodium through the pump (“digitalis hypothesis”). CTS possess three structural components: a saturated/unsaturated lactone ring, steroid core, and sugar moiety, each of which may be involved in NKP inhibition/stimulation. It is now known that inhibition of the NKP in patients with HF increases mortality, and all major beneficial treatments increase its activity. Endogenous circulating CTS such as ouabain are generally thought to inhibit the NKP, despite studies sporadically reporting ouabain-induced pump stimulation. This study aims to identify whether ouabain-induced pump stimulation occurs, and if so, which structural components are involved in causing pump stimulation. Methods & Results: Cardiac myocytes were isolated from male New Zealand White rabbits, placed in a Tyrode’s solution, and whole-cell patch clamped. They were exposed to 0-30nM ouabain, 0-50nM dihydroouabain (ouabain with a saturated lactone ring) or 0-500nM ouabagenin (ouabain lacking a sugar moiety) for 1 min, followed by a potassium-free solution, with the difference in current yielding the NKP current. Compared to the 0.47±0.05 pA/pF Tyrode’s solution control (n=11), 5nM ouabain significantly increased NKP current to 0.69±0.09 pA/pF ( P <0.05, n=6). Exposure to dihydroouabain or ouabagenin did not significantly change NKP current in the studied concentration range. Cell viability assays carried out on the breast cancer cell line MCF7, which have an NKP structure extremely similar to that of cardiomyocytes, showed significantly elevated viability above control values (n=2) following 24h treatment with 0-9nM ouabain; maximum viability was 116±5% at 0.28nM ( P <0.05, n=4). A significant change in viability was not observed for ouabagenin or digoxin in the same concentration range. Conclusion: Low-dose ouabain uniquely stimulates NKP activity. Low-dose dihydroouabain and ouabagenin do not, suggesting that a sugar moiety and unsaturated lactone ring are required for pump stimulation. Ouabain in its unaltered form may be a potential treatment for HF.

Abstract 379: Silencing of Fxyd1 Uncouples Enos in Human Umbilical Vein Endothelial Cells

Introduction/Aims: FXYD1 has been shown to be a master regulator of the sodium potassium pump activity in the caveolae of heart tissue, protecting the pump from glutathionylation and oxidative inhibition. However, very little is known about its expression and function in the endothelium, particularly in regard to its potential interaction with other caveolae proteins. Given the molecular mechanism of eNOS uncoupling is via glutathionylation, we examined whether FXYD1 was expressed in endothelial cells, and whether it had a functional partnership with eNOS. Methods: Co-immunoprecipitation was used to study the interaction of FXYD1 and eNOS. GSH antibody was used for immunodetection of eNOS glutathionylation. Silencing of FXYD1 using siRNA followed by DAF staining or spin trap were used to study the effects of FXYD1 on eNOS function. Enzyme-linked immunosorbent assay was also employed to quantify eNOS glutathionylation level in human erythrocyte samples post exposure to angiotensin II (0 to 500 nM) ex vivo. Results: FXYD1 co-immunoprecipitated with eNOS in human umbilical vein endothelial cells (HUVECs). Silencing of FXYD1 resulted in significantly increased eNOS glutathionylation, associated with significantly reduced NO bioavailability under baseline and acetylcholine stimulated conditions (1μM, 20mins). Furthermore, a dose-dependent increase in eNOS glutathionylation level was also observed in human erythrocyte exposed to angiotensin II. Discussion: Our findings demonstrated a novel functional partnership of FXYD1 with eNOS, protecting this vital enzyme from glutathionylation-mediated uncoupling. This has important implications for our understanding of ROS-signalling in the vasculature. Furthermore, FXYD1 expression/eNOS glutathionylation may serve as important biomarkers for oxidative stress.

Na/K pump regulation of cardiac repolarization: insights from a systems biology approach

The sodium-potassium pump is widely recognized as the principal mechanism for active ion transport across the cellular membrane of cardiac tissue, being responsible for the creation and maintenance of the transarcolemmal sodium and potassium gradients, crucial for cardiac cell electrophysiology. Importantly, sodium-potassium pump activity is impaired in a number of major diseased conditions, including ischemia and heart failure. However, its subtle ways of action on cardiac electrophysiology, both directly through its electrogenic nature and indirectly via the regulation of cell homeostasis, make it hard to predict the electrophysiological consequences of reduced sodium-potassium pump activity in cardiac repolarization. In this review, we discuss how recent studies adopting the systems biology approach, through the integration of experimental and modeling methodologies, have identified the sodium-potassium pump as one of the most important ionic mechanisms in regulating key properties of cardiac repolarization and its rate dependence, from subcellular to whole organ levels. These include the role of the pump in the biphasic modulation of cellular repolarization and refractoriness, the rate control of intracellular sodium and calcium dynamics and therefore of the adaptation of repolarization to changes in heart rate, as well as its importance in regulating pro-arrhythmic substrates through modulation of dispersion of repolarization and restitution. Theoretical findings are consistent across a variety of cell types and species including human, and widely in agreement with experimental findings. The novel insights and hypotheses on the role of the pump in cardiac electrophysiology obtained through this integrative approach could eventually lead to novel therapeutic and diagnostic strategies.

Plantar cutaneous sensitivity as a risk for falls in the elderly

The risk factors for falls in the elderly are many and may be due to syncope and pre-syncope caused by cardiovascular diseases; neurological diseases, such as epilepsy and Parkinson’s disease; dementia disorders; and dependence in activities of daily life 1 . Other risk factors that also cause syncope and fall include the adverse effect or inappropriate use of several concurrent medications, drugs which are central nervous system-depressant, or act on the cardiocirculatory system 1 . Systemic diseases combined with sensorineural degeneration and neuromuscular disorders due to aging accentuate the risk of falling, particularly in the frail elderly. Vertigo and imbalance are also frequent causes of falls, often associated with metabolic disorders, mainly uncontrolled diabetes without the well-known clinical manifestations of the disease 2,3 . Diabetes leads to glucose fluctuations in metabolically active structures of the inner ear, interfering with the sodiumpotassium pump activity. This pump creates the electrical potentials of the cochlear and vestibular neuroepithelial cells that, when altered, produce auditory and vestibular signs and symptoms. Among the causes of change in body balance there are also the reduced number of mechanoreceptors located in the feet, and the increase in the vibrational excitation threshold of the plantar cutaneous mechanoreceptors due to aging. Plantar sensitivity is an important source of information for balance control, as it codifies the changes in pressure under the feet, especially during gait. This information reaches the brain, which senses the body position and, if necessary, generates postural reflexes to maintain an upright position 4 . This sensitivity loss may be due to several diseases, particularly diabetes 5 . About 50% of diabetic patients over 60 years of age have this disorder, with or without complaint. Moreover, even the healthy elderly may have subclinical neuropathy. It has been widely demonstrated that this loss plays an important role in balance disorders in the elderly population 4-6 . Change in plantar cutaneous sensitivity is an independent predictor of falls; 30% of the elderly people aged 65, and 40% of those aged 75 and over have fallen at least once 7 . Two or more falls in six months significantly increases the risk of falls in the frail elderly. Quality of life is compromised by a sense of instability that causes fear of falling, especially if the elder has fallen recently 7 . Fear of falling leads to reduced mobility, increasing sedentary lifestyle and creating a vicious cycle that further increases the risk 7,8 . Frequent falls often

An Interesting Link Between Microgravity and Psychiatric Diseases

Back to table of contents Previous article Next article LETTERFull AccessAn Interesting Link Between Microgravity and Psychiatric DiseasesBurak Yulug,Gokhan Erkol,Ertugrul Kilic,Burak YulugSearch for more papers by this author,Gokhan ErkolSearch for more papers by this author,Ertugrul KilicSearch for more papers by this author,Published Online:1 Oct 2010https://doi.org/10.1176/jnp.2010.22.4.451.e12AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail To the Editor: The exposure to altered microgravity during space travel has been shown to induce changes in the limbic system which are reflected in many of the physical behaviors seen in the astronauts. Although the neurobiological basis of this phenomenon is still obscure, several psychiatric issues including anxiety, depression, psychosis, psychosomatic symptoms, emotional problems, and cognitive changes have been observed after spaceflight. 1 However, studies evaluating the impact of a microgravity environment on brain function revealed significant CNS changes, and the direct effect of microgravity has already been shown by in vivo and in vitro animal studies. 2 It has recently been shown that microgravity caused an oxidative imbalance in the hypothalamus suggesting an organic interaction between the environment and the hypothalamic-pituitary-adrenal axis. 3 This was suggested by recent animal and human studies showing significant increases of prolactin, cortisol, and ACTH levels after exposure to the microgravity environment. This concurs with recent studies showing a strong and significant correlation between circulating stress hormones, psychological status, and transient metabolic changes on the special limbic and cortical regions in humans. Another study evaluating the neurotransmitter receptor binding and NA+, K+-ATPase activity in the limbic and cortical regions after exposure to a microgravity environment showed significantly increased 5-HT 1 receptor number in the hippocampus in contrast to a significantly depressed cortical sodium-potassium pump activity. 4 This could be related to mechanisms of structural adaptation to a decreased afferent flow in the hemisphere cortex in microgravity. This was also suggested by studies indicating inhibited cortical function in simulated microgravity conditions. These findings are interesting in light of increasing evidence regarding the functional metabolic interaction between the cortex and the limbic system by the normal regulation of mood, the development of psychiatric diseases, and the recovery from depression after treatment. Additionally, the decreased afferent flow to the hemisphere cortex can arise as a result of sensory deprivation, which can be considered an unenriched environment. We can hypothesize that cortisol-related decreases in cortical metabolism can be a risk factor for the development of psychiatric diseases, such as hippocampal damage, which has also been shown to predispose depression. An interesting animal study revealed that animals exposed to an unenriched environment are more likely to behave like depressive animals. This is supported by studies showing that an enriched environment is capable of reversing the stress-induced disturbances and enhancing the neurogenesis. 5This finding suggests that weightlessness may affect adaptational processes not only due to weightlessness but also due to exposure to other stressors, which can mediate the psychobiological effects of the altered gravity experienced by microgravity conditions. However, the detailed underlying pathophysiological mechanism of such a possible link between stress, microgravity, and psychiatric diseases should be confirmed with further neuroimaging studies.University of Regensburg, Department of Neurology, Regensburg, GermanyUniversity of Istanbul, Cerrahpasa Medical School, Department of Neurology, Istanbul, TurkeyBrain Research Laboratory, University of Yeditepe, Istanbul

Cleistanthus collinus induces type I distal renal tubular acidosis and type II respiratory failure in rats

Background and Purpose:A water decoction of the poisonous shrub Cleistanthus collinus is used for suicidal purposes. The mortality rate is 28%. The clinical profile includes distal renal tubular acidosis (DRTA) and respiratory failure. The mechanism of toxicity is unclear.Objectives:To demonstrate features of C. collinus toxicity in a rat model and to identify its mechanism(s) of action.Materials and Methods:Rats were anesthetized and the carotid artery was cannulated. Electrocardiogram and respiratory movements were recorded. Either aqueous extract of C. collinus or control solution was administered intraperitoneally. Serial measurements of blood gases, electrolytes and urinary pH were made. Isolated brush border and basolateral membranes from rat kidney were incubated with C. collinus extract and reduction in ATPase activity was assessed. Venous blood samples from human volunteers and rats were incubated with an acetone extract of C. collinus and plasma potassium was estimated as an assay for sodium–potassium pump activity.Results:The mortality was 100% in tests and 17% in controls. Terminal event in test animals was respiratory arrest. Controls had metabolic acidosis, respiratory compensation acidic urine and hyperkalemia. Test animals showed respiratory acidosis, alkaline urine and low blood potassium as compared to controls. C. collinus extract inhibited ATPase activity in rat kidney. Plasma K+ did not increase in human blood incubated with C. collinus extract.Conclusions and Implications:Active principles of C. collinus inhibit proton pumps in the renal brush border, resulting in type I DRTA in rats. There is no inhibition of sodium–potassium pump activity. Test animals develop respiratory acidosis, and the immediate cause of death is respiratory arrest.

Actions of antidiuretic hormone analogues on intact and nystatin‐permeabilized frog skins

The roles of two antidiuretic hormone analogues, namely arginine vasotocin (AVT) and lysine vasopressin (LVP), in solute transport across the ventral abdominal skin of frogs (Rana hexadactyla) were studied using voltage-clamp methods on intact and nystatin-permeabilized preparations. Arginine vasotocin (40 nm), the amphibian analogue of antidiuretic hormone, did not have any effect on the skin of Rana hexadactyla. However, LVP, the porcine antidiuretic hormone, increased the transepithelial potential difference (TEPD) and short-circuit current (SCC) significantly, without affecting the slope conductance. Lysine vasopressin had no action subsequent to addition of amiloride (100 microm) on the apical side or ouabain (10 microm) on the basolateral side. Lysine vasopressin increased slope conductance in the nystatin-permeablized skin while decreasing TEPD. Such a change was not seen in chloride-free solutions. To elucidate the mechanism of action of LVP on intact skin, experiments were done with forskolin and a V(2) receptor blocker. The effects of forskolin (10 microm) were different from those of LVP in that forskolin significantly increased SCC and conductance of the intact skin, while decreasing TEPD. The forskolin-induced increase in conductance was not abolished by amiloride. Use of the V(2) receptor blocker inhibited the effects of LVP. We conclude that AVT does not have an action on the skin of Rana hexadactyla. Lysine vasopressin enhances transepithelial sodium transport by increasing sodium-potassium pump activity, while not affecting the epithelial sodium channel conductance. Lysine vasopressin also enhances an inward-directed conductance on the basolateral membrane, probably a chloride conductance. The action of LVP on the intact frog skin is through the V(2) receptors; however, downstream signalling does not seem to be mediated by cAMP. Analysis of the electrophysiological model of frog skin with LVP allows us additionally to conclude that modulation of channel activity and not carrier-mediated transport affects slope conductance.

Heat Stress and Hormetin-Induced Hormesis in Human Cells: Effects on Aging, Wound Healing, Angiogenesis, and Differentiation

Accumulation of molecular damage and increased molecular heterogeneity are hallmarks of cellular aging. Mild stress-induced hormesis can be an effective way for reducing the accumulation of molecular damage, and thus slowing down aging from within. We have shown that repeated mild heat stress (RMHS) has anti-aging effects on growth and various other cellular and biochemical characteristics of normal human skin fibroblasts and keratinocytes undergoing aging in vitro. RMHS given to human cells increased the basal levels of various chaperones, reduced the accumulation of damaged proteins, stimulated proteasomal activities, increased the cellular resistance to other stresses, enhanced the levels of various antioxidant enzymes, enhanced the activity and amounts of sodium-potassium pump, and increased the phosphorylation-mediated activities of various stress kinases. We have now observed novel hormetic effects of mild heat stress on improving the wound healing capacity of skin fibroblasts and on enhancing the angiogenic ability of endothelial cells. We have also tested potential hormetins, such as curcumin and rosmarinic acid in bringing about their beneficial effects in human cells by inducing stress response pathways involving heat shock proteins and hemeoxygenase HO-1. These data further support the view that mild stress-induced hormesis can be applied for the modulation, intervention and prevention of aging and age-related impairments.

Delivery of exogenous ATP maintains sodium-potassium pump activity in endothelial cells during chemical hypoxia

Maldonado, Claudio PhD; Grossi, Federico V. MD; Khan, Mashiur BS; Soni, Chirag BS; Okafor, Mansim C. PhD; Vasilic, Dalibor MD; Ehringer, William C. PhD; Perez-Abadia, Gustavo MD; Delamere, Nicholas A. PhD; Barker, John H. MD Author Information

Canrenone competes for the same site of ouabain on Na+/K+-ATPase

Recently several studies suggested the existence in human plasma of an endogenous digitalis-like factors (EDLF) similar to ouabain which could have a role in the pathogenesis of some forms of hypertension. Canrenone is a metabolic product of the antialdosteronic drug spironolactone, used in hypertensive therapy and recently described as a blocker of ouabain effects. The aim of this study was to evaluate the effect of canrenone on Na+/K+-ATPase in relation to ouabain investigating its interaction with the ouabain receptor and with the sodium-potassium pump activity. For this purpose we employed a3H ouabain receptor assay in human placental membranes and a 86Rb uptake assay in human erythrocytes. Increasing concentration of canrenone (0-180 μM) partially inhibited the ouabain sensitive 86Rb uptake in red blood cells until 40%. Moreover canrenone partially competed for 30% with 3H ouabain binding in placental membrane receptors in absence and in presence of 5 mM K+, respectively at a concentration of 350 μM and 70 μM. When the displacement of ouabain (10-7M) by canrenone (180 μM) was valuated, measuring ouabain sensitive 86Rb uptake, a recovery of 86Rb uptake was obtained only in presence of 5 mM K+. Finally Scatchard plot from radioreceptor assay showed that ouabain in absence and presence of canrenone intersected at a common point on the abscissal axis. These results provide evidence that canrenone is a partial competitive agonist of ouabain and interacts with the same receptor site suggesting that it could be used in hypertensive states with high levels of endogenous ouabain.

Tissue-specific modulation of Na, K-ATPase α-subunit gene expression in uremic rats

Chronic renal failure in the rat is associated with an impaired extrarenal potassium handling, whereas a renal adaptive mechanism of the remaining nephrons has been described. To understand the molecular basis of potassium homeostasis during renal failure we investigated the in vitro pump activity and the catalytic mRNA transcription in three different tissues: skeletal muscle, isolated adipocytes and kidney. The activity of the sodium pump, as measured by ouabain-sensitive 86Rb/K uptake in isolated adipocytes and skeletal muscle fibers, revealed a significant reduction of the pump activity in uremic rats. The reduction of the Na, K-ATPase activity in adipose tissue was associated with a similar decrement of both catalytic subunits (alpha 1 and alpha 2), whereas in the skeletal muscle tissue was only related to a decrease in the activity of the alpha 1 isoform. The expression of rat Na, K-ATPase catalytic isoforms mRNAs in kidney, muscle and adipose tissue from control and chronic renal failure rats was investigated at the molecular level with cDNA probes specific for the catalytic isoforms (alpha 1 and alpha 2). Northern blot analysis revealed that the respective catalytic mRNAs of uremic rats are regulated in a tissue-specific manner that are in agreement with the sodium-potassium pump activity. Muscle and adipose tissue showed a decrement in the levels of expression for the alpha 1 isoform mRNA. In contrast to these tissues, an increment in alpha 1 mRNA expression was observed in the kidney of rats with chronic renal failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen peroxide stimulates sodium-potassium pump activity in cultured pulmonary arterial endothelial cells.

Oxidant injury to pulmonary vascular endothelium is an important factor in the pathogenesis of acute lung injury. Oxidant injury to other cell types has been reported to alter the function of Na-K-adenosinetriphophatase (ATPase) an enzyme important in maintenance of cellular ionic homeostasis and in transport of ions across biological membranes. We investigated the effect of H2O2 (0.001-10 mM) or xanthine (X) (15.2 micrograms/ml) plus xanthine oxidase (XO) (0.0153 U/ml) on the Na-K pump activity of cultured bovine pulmonary arterial endothelial cells (PAECs). We used a functional assay, using 86RbCl as a tracer for K+ and expressing Na-K pump activity as ouabain-inhibitable K+ uptake. Our results demonstrate that H2O2 and X/XO stimulate Na-K pump activity of bovine PAECs, an effect prevented by catalase. In addition, we assessed the affinity, number, and turnover of [3H]ouabain binding sites on intact endothelial monolayers and found that H2O2 increased affinity to [3H]ouabain, decreased the number of binding sites, and increased the rate of pump turnover. Influx of 22Na increased in response to a nonlytic concentration of H2O2. Cell injury, as assessed by 51Cr release, adherent cell number, and phase-microscopic morphology, was not observed after 30-min incubations with the lowest dose (1 mM) of H2O2 effective in stimulating Na-K pump activity, or after incubation with X/XO. Na-K pump inhibition by ouabain significantly increased the 51Cr release caused by H2O2 or by X/XO, suggesting that the increase in Na-K pump activity may be a compensatory response to the cellular alterations produced by H2O2. Incubation with H2O2 decreased cell ATP content, an effect which was not prevented by coincubation with ouabain. In summary, these results show that H2O2 increases Na-K pump activity of PAECs, an effect mediated, at least in part, by increased intracellular [Na] and by an increased rate of pump turnover. It is possible that the increased pump activity may be an early marker of endothelial cell perturbation.

Sodium-potassium pump activity contributes to the age-related decline in resting metabolic rate.

We examined the hypothesis that a decline in Na-K pump activity contributes to the lower resting metabolic rate (RMR) in older males, independent of the loss of fat-free mass. Plasma and erythrocyte Na and K concentrations were measured using flame photometry. Changes in these concentrations after incubation with and without ouabain were used to calculate erythrocyte Na-K pump rate and rate constant in 27 younger (28 +/- 7 yr) and 25 older (67 +/- 6 yr) men. Older men showed an 18% lower erythrocyte Na-K pump rate constant (0.32 +/- 0.09 vs. 0.39 +/- 0.10 h-1; P < 0.01), a 12% lower RMR (1.10 +/- 0.14 vs. 1.25 +/- 0.14 kcal/min; P < 0.01) and an 8% lower level of fat-free mass (61.7 +/- 5.8 vs. 67.4 +/- 8.1 kg; P < 0.01) relative to younger men. A lower RMR persisted in older men (1.14 +/- 0.12 kcal/min) compared to younger men (1.21 +/- 0.12 kcal/min; P < 0.05) after control for the effect of fat-free mass. No differences in RMR were found, however, between older (1.17 +/- 0.13 kcal/min) and younger men (1.20 +/- 0.13 kcal/min) after controlling for both fat-free mass and the erythrocyte Na-K pump rate constant. A positive relation was noted between RMR and the erythrocyte Na-K pump rate constant, after removing the effects of fat-free mass (partial r = 0.30; P < 0.05). Our results support the conclusions that: 1) the in vivo activity of the Na-K pump is related to RMR, and 2) the age-related reduction in Na-K pump activity is a partial contributor to the decline in RMR in older men.

Sodium-potassium pump activity contributes to the age-related decline in resting metabolic rate

Sodium-potassium pump activity contributes to the age-related decline in resting metabolic rate