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 (10 s) or repeated tetani (60 Hz for 200 ms, every 2 s, for 30 s or 300 s) 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 -71 mV (surface fibres were more depolarised), and [Na+]i was 14 mM. With deeper fibres, 10 s stimulation (2-150 Hz) increased [Na+]i to 38-46 mM whilst simultaneously causing hyperpolarisations (7.3 mV for 2-90 Hz). Fatiguing stimulation for 30 s or 300 s led to end-stimulation resting EM of -61 to -53 mV, which recovered rapidly (T1/2, 8-22 s). Mean end-stimulation [Na+]i increased to 86-101 mM with both fatigue protocols and the [Na+]i recovery time-course (T1/2, 21-35 s) 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 10 s 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.

Na,K-ATPase: A murzyme facilitating thermodynamic equilibriums at the membrane-interface.

The redox metabolic paradigm of murburn concept advocates that diffusible reactive species (DRS, particularly oxygen-centric radicals) are mainstays of physiology, and not mere pathological manifestations. The murburn purview of cellular function also integrates the essential principles of bioenergetics, thermogenesis, homeostasis, electrophysiology, and coherence. In this context, any enzyme that generates/modulates/utilizes/sustains DRS functionality is called a murzyme. We have demonstrated that several water-soluble (peroxidases, lactate dehydrogenase, hemogoblin, etc.) and membrane-embedded (Complexes I-V in mitochondria, Photosystems I/II in chloroplasts, rhodopsin/transducin in rod cells, etc.) proteins serve as murzymes. The membrane protein of Na,K-ATPase (NKA, also known as sodium-potassium pump) is the focus of this article, owing to its centrality in neuro-cardio-musculo electrophysiology. Herein, via a series of critical queries starting from the geometric/spatio-temporal considerations of diffusion/mass transfer of solutes in cells to an update on structural/distributional features of NKA in diverse cellular systems, and from various mechanistic aspects of ion-transport (thermodynamics, osmoregulation, evolutionary dictates, etc.) to assays/explanations of inhibitory principles like cardiotonic steroids (CTS), we first highlight some unresolved problems in the field. Thereafter, we propose and apply a minimalist murburn model of trans-membrane ion-differentiation by NKA to address the physiological inhibitory effects of trans-dermal peptide, lithium ion, volatile anesthetics, confirmed interfacial DRS + proton modulators like nitrophenolics and unsaturated fatty acid, and the diverse classes of molecules like CTS, arginine, oximes, etc. These explanations find a pan-systemic connectivity with the inhibitions/uncouplings of other membrane proteins in cells.

Genome sequence of Ophryocystis elektroscirrha, an apicomplexan parasite of monarch butterflies: cryptic diversity and response to host-sequestered plant chemicals.

Apicomplexa are ancient and diverse organisms which have been poorly characterized by modern genomics. To better understand the evolution and diversity of these single-celled eukaryotes, we sequenced the genome of Ophryocystis elektroscirrha, a parasite of monarch butterflies, Danaus plexippus. We contextualize our newly generated resources within apicomplexan genomics before answering longstanding questions specific to this host-parasite system. To start, the genome is miniscule, totaling only 9 million bases and containing fewer than 3,000 genes, half the gene content of two other sequenced invertebrate-infecting apicomplexans, Porospora gigantea and Gregarina niphandrodes. We found that O. elektroscirrha shares different orthologs with each sequenced relative, suggesting the true set of universally conserved apicomplexan genes is very small indeed. Next, we show that sequencing data from other potential host butterflies can be used to diagnose infection status as well as to study diversity of parasite sequences. We recovered a similarly sized parasite genome from another butterfly, Danaus chrysippus, that was highly diverged from the O. elektroscirrha reference, possibly representing a distinct species. Using these two new genomes, we investigated potential evolutionary response by parasites to toxic phytochemicals their hosts ingest and sequester. Monarch butterflies are well-known to tolerate toxic cardenolides thanks to changes in the sequence of their Type II ATPase sodium pumps. We show that Ophryocystis completely lacks Type II or Type 4 sodium pumps, and related proteins PMCA calcium pumps show extreme sequence divergence compared to other Apicomplexa, demonstrating new avenues of research opened by genome sequencing of non-model Apicomplexa.

The vascular Na,K-ATPase: clinical implications in stroke, migraine, and hypertension.

In the vascular wall, the Na,K-ATPase plays an important role in the control of arterial tone. Through cSrc signaling, it contributes to the modulation of Ca2+ sensitivity in vascular smooth muscle cells. This review focuses on the potential implication of Na,K-ATPase-dependent intracellular signaling pathways in severe vascular disorders; ischemic stroke, familial migraine, and arterial hypertension. We propose similarity in the detrimental Na,K-ATPase-dependent signaling seen in these pathological conditions. The review includes a retrospective proteomics analysis investigating temporal changes after ischemic stroke. The analysis revealed that the expression of Na,K-ATPase α isoforms is down-regulated in the days and weeks following reperfusion, while downstream Na,K-ATPase-dependent cSrc kinase is up-regulated. These results are important since previous studies have linked the Na,K-ATPase-dependent cSrc signaling to futile recanalization and vasospasm after stroke. The review also explores a link between the Na,K-ATPase and migraine with aura, as reduced expression or pharmacological inhibition of the Na,K-ATPase leads to cSrc kinase signaling up-regulation and cerebral hypoperfusion. The review discusses the role of an endogenous cardiotonic steroid-like compound, ouabain, which binds to the Na,K-ATPase and initiates the intracellular cSrc signaling, in the pathophysiology of arterial hypertension. Currently, our understanding of the precise control mechanisms governing the Na,K-ATPase/cSrc kinase regulation in the vascular wall is limited. Understanding the role of vascular Na,K-ATPase signaling is essential for developing targeted treatments for cerebrovascular disorders and hypertension, as the Na,K-ATPase is implicated in the pathogenesis of these conditions and may contribute to their comorbidity.

Compound-Specific Behavioral and Enzymatic Resistance to Toxic Milkweed Cardenolides in a Generalist Bumblebee Pollinator.

Plant secondary metabolites that defend leaves from herbivores also occur in floral nectar. While specialist herbivores often have adaptations providing resistance to these compounds in leaves, many social insect pollinators are generalists, and therefore are not expected to be as resistant to such compounds. The milkweeds, Asclepias spp., contain toxic cardenolides in all tissues including floral nectar. We compared the concentrations and identities of cardenolides between tissues of the North American common milkweed Asclepias syriaca, and then studied the effect of the predominant cardenolide in nectar, glycosylated aspecioside, on an abundant pollinator. We show that a generalist bumblebee, Bombus impatiens, a common pollinator in eastern North America, consumes less nectar with experimental addition of ouabain (a standard cardenolide derived from Apocynacid plants native to east Africa) but not with addition of glycosylated aspecioside from milkweeds. At a concentration matching that of the maximum in the natural range, both cardenolides reduced activity levels of bees after four days of consumption, demonstrating toxicity despite variation in behavioral deterrence (i.e., consumption). In vitro enzymatic assays of Na+/K+-ATPase, the target site of cardenolides, showed lower toxicity of the milkweed cardenolide than ouabain for B. impatiens, indicating that the lower deterrence may be due to greater tolerance to glycosylated aspecioside. In contrast, there was no difference between the two cardenolides in toxicity to the Na+/K+-ATPase from a control insect, the fruit fly Drosophila melanogaster. Accordingly, this work reveals that even generalist pollinators such as B. impatiens may have adaptations to reduce the toxicity of specific plant secondary metabolites that occur in nectar, despite visiting flowers from a wide variety of plants over the colony's lifespan.

Oral digoxin effects on exercise performance, K+ regulation and skeletal muscle Na+ ,K+ -ATPase in healthy humans.

We investigated whether digoxin lowered muscle Na+ ,K+ -ATPase (NKA), impaired muscle performance and exacerbated exercise K+ disturbances. Ten healthy adults ingested digoxin (0.25 mg; DIG) or placebo (CON) for 14 days and performed quadriceps strength and fatiguability, finger flexion (FF, 105%peak-workrate , 3 × 1 min, fourth bout to fatigue) and leg cycling (LC, 10 min at 33% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ and 67% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ , 90% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ to fatigue) trials using a double-blind, crossover, randomised, counter-balanced design. Arterial (a) and antecubital venous (v) blood was sampled (FF, LC) and muscle biopsied (LC, rest, 67% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ , fatigue, 3 h after exercise). In DIG, in resting muscle, [3 H]-ouabain binding site content (OB-Fab ) was unchanged; however, bound-digoxin removal with Digibind revealed total ouabain binding (OB+Fab ) increased (8.2%, P = 0.047), indicating 7.6% NKA-digoxin occupancy. Quadriceps muscle strength declined in DIG (-4.3%, P = 0.010) but fatiguability was unchanged. During LC, in DIG (main effects), time to fatigue and [K+ ]a were unchanged, whilst [K+ ]v was lower (P = 0.042) and [K+ ]a-v greater (P = 0.004) than in CON; with exercise (main effects), muscle OB-Fab was increased at 67% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ (per wet-weight, P = 0.005; per protein P = 0.001) and at fatigue (per protein, P = 0.003), whilst [K+ ]a , [K+ ]v and [K+ ]a-v were each increased at fatigue (P = 0.001). During FF, in DIG (main effects), time to fatigue, [K+ ]a , [K+ ]v and [K+ ]a-v were unchanged; with exercise (main effects), plasma [K+ ]a , [K+ ]v , [K+ ]a-v and muscle K+ efflux were all increased at fatigue (P = 0.001). Thus, muscle strength declined, but functional muscle NKA content was preserved during DIG, despite elevated plasma digoxin and muscle NKA-digoxin occupancy, with K+ disturbances and fatiguability unchanged. KEY POINTS: The Na+ ,K+ -ATPase (NKA) is vital in regulating skeletal muscle extracellular potassium concentration ([K+ ]), excitability and plasma [K+ ] and thereby also in modulating fatigue during intense contractions. NKA is inhibited by digoxin, which in cardiac patients lowers muscle functional NKA content ([3 H]-ouabain binding) and exacerbates K+ disturbances during exercise. In healthy adults, we found that digoxin at clinical levels surprisingly did not reduce functional muscle NKA content, whilst digoxin removal by Digibind antibody revealed an ∼8% increased muscle total NKA content. Accordingly, digoxin did not exacerbate arterial plasma [K+ ] disturbances or worsen fatigue during intense exercise, although quadriceps muscle strength was reduced. Thus, digoxin treatment in healthy participants elevated serum digoxin, but muscle functional NKA content was preserved, whilst K+ disturbances and fatigue with intense exercise were unchanged. This resilience to digoxin NKA inhibition is consistent with the importance of NKA in preserving K+ regulation and muscle function.

Selective electrical stimulation of low versus high diameter myelinated fibers and its application in pain relief: a modeling study.

Electrical stimulation of peripheral nerve fibers has always been an attractive field of research. Due to the higher activation threshold, the stimulation of small fibers is accompanied by the stimulation of larger ones. It is therefore necessary to design a specific stimulation theme in order to only activate narrow fibers. There is evidence that stimulating Aδ fibers can activate endogenous pain-relieving mechanisms. However, both selective stimulation and reducing pain by activating small nociceptive fibers are still poorly investigated. In this study, using high-frequency stimulation waveforms (5-20 kHz), computational modeling provides a simple framework for activating narrow nociceptive fibers. Additionally, a model of myelinated nerve fibers is modified by including sodium-potassium pump and investigating its effects on neuronal stimulation. Besides, a modified mathematical model of pain processing circuits in the dorsal horn is presented that consists of supraspinal pain control mechanisms. Hence, by employing this pain-modulating model, the mechanism of the reduction of pain by activating nociceptive fibers is explored. In the case of two fibers with the same distance from the point source electrode, a single stimulation waveform is capable of blocking one large fiber and stimulating another small fiber. Noteworthy, the Na/K pump model demonstrated that it does not have a significant effect on the activation threshold and firing frequency of fiber. Ultimately, results suggest that the descending pathways of Locus coeruleus may effectively contribute to pain relief through stimulation of nociceptive fibers, which will be beneficial for clinical interventions.

Molecular mechanisms of local adaptation for salt-tolerance in a treefrog.

Salinization is a global phenomenon affecting ecosystems and forcing freshwater organisms to deal with increasing levels of ionic stress. However, our understanding of mechanisms that permit salt tolerance in amphibians is limited. This study investigates mechanisms of salt tolerance in locally adapted, coastal populations of a treefrog, Hyla cinerea. Using a common garden experiment, we (i) determine the extent that environment (i.e., embryonic and larval saltwater exposure) or genotype (i.e., coastal vs. inland) affects developmental benchmarks and transcriptome expression, and (ii) identify genes that may underpin differences in saltwater tolerance. Differences in gene expression, survival, and plasma osmolality were most strongly associated with genotype. Population genetic analyses on expressed genes also delineated coastal and inland groups based on genetic similarity. Coastal populations differentially expressed osmoregulatory genes including ion transporters (atp1b1, atp6V1g2, slc26a), cellular adhesion components (cdh26, cldn1, gjb3, ocln), and cytoskeletal components (odc1-a, tgm3). Several of these genes are the same genes expressed by euryhaline fish after exposure to freshwater, which is a novel finding for North American amphibians and suggests that these genes may be associated with local salinity adaptation. Coastal populations also highly expressed glycerol-3-phosphate dehydrogenase 1 (gpd1), which indicates they use glycerol as a compatible osmolyte to reduce water loss - another mechanism of saltwater tolerance previously unknown in frogs. These data signify that Hyla cinerea inhabiting coastal, brackish wetlands have evolved a salt-tolerant ecotype, and highlights novel candidate pathways that can lead to salt tolerance in freshwater organisms facing habitat salinization.

Intracellular Na+ Modulates Pacemaking Activity in Murine Sinoatrial Node Myocytes: An In Silico Analysis.

Background: The mechanisms underlying dysfunction in the sinoatrial node (SAN), the heart's primary pacemaker, are incompletely understood. Electrical and Ca2+-handling remodeling have been implicated in SAN dysfunction associated with heart failure, aging, and diabetes. Cardiomyocyte [Na+]i is also elevated in these diseases, where it contributes to arrhythmogenesis. Here, we sought to investigate the largely unexplored role of Na+ homeostasis in SAN pacemaking and test whether [Na+]i dysregulation may contribute to SAN dysfunction. Methods: We developed a dataset-specific computational model of the murine SAN myocyte and simulated alterations in the major processes of Na+ entry (Na+/Ca2+ exchanger, NCX) and removal (Na+/K+ ATPase, NKA). Results: We found that changes in intracellular Na+ homeostatic processes dynamically regulate SAN electrophysiology. Mild reductions in NKA and NCX function increase myocyte firing rate, whereas a stronger reduction causes bursting activity and loss of automaticity. These pathologic phenotypes mimic those observed experimentally in NCX- and ankyrin-B-deficient mice due to altered feedback between the Ca2+ and membrane potential clocks underlying SAN firing. Conclusions: Our study generates new testable predictions and insight linking Na+ homeostasis to Ca2+ handling and membrane potential dynamics in SAN myocytes that may advance our understanding of SAN (dys)function.

Na+/K+-ATPase Revisited: On Its Mechanism of Action, Role in Cancer, and Activity Modulation.

Maintenance of Na+ and K+ gradients across the cell plasma membrane is an essential process for mammalian cell survival. An enzyme responsible for this process, sodium-potassium ATPase (NKA), has been currently extensively studied as a potential anticancer target, especially in lung cancer and glioblastoma. To date, many NKA inhibitors, mainly of natural origin from the family of cardiac steroids (CSs), have been reported and extensively studied. Interestingly, upon CS binding to NKA at nontoxic doses, the role of NKA as a receptor is activated and intracellular signaling is triggered, upon which cancer cell death occurs, which lies in the expression of different NKA isoforms than in healthy cells. Two major CSs, digoxin and digitoxin, originally used for the treatment of cardiac arrhythmias, are also being tested for another indication-cancer. Such drug repositioning has a big advantage in smoother approval processes. Besides this, novel CS derivatives with improved performance are being developed and evaluated in combination therapy. This article deals with the NKA structure, mechanism of action, activity modulation, and its most important inhibitors, some of which could serve not only as a powerful tool to combat cancer, but also help to decipher the so-far poorly understood NKA regulation.

Inhibition of glycogenolysis prolongs action potential repriming period and impairs muscle function in rat skeletal muscle.

KEY POINTS: Muscle glycogen content is associated with muscle function, but the physiological link between the two is poorly understood. This study investigated the effects of inhibiting glycogenolysis, while maintaining high overall energy status, on different aspects of muscle function. We demonstrate here that Na+ ,K+ -ATPase activity depends on glycogenolytically derived ATP regardless of high global ATP, with a decrease in activity leading to reduced force production and accelerated fatigue development. The results support the concept of compartmentalized energy transfer with glycogen metabolism playing a crucial role in intramuscular ATP resynthesis and ion regulation. This study gives specific insights into muscular function and may help towards a better understanding of glycogen storage diseases and muscle fatigue. ABSTRACT: Skeletal muscle glycogen content is associated with muscle function and fatigability. However, little is known about the physiological link between glycogen content and muscle function. Here we aimed to investigate the importance of glycogenolytically derived ATP per se on muscle force and action potential (AP) repriming period, i.e. the time before a second AP can be produced (indicative of Na+ ,K+ -ATPase activity). Single fibres from rat extensor digitorum longus muscles were isolated and mechanically skinned in order to investigate force production and the AP repriming period while global ATP and PCr concentrations were kept high. The importance of glycogenolytically derived ATP was studied by inhibition of glycogen phosphorylase (1,4-dideoxy-1,4-imino-d-arabinitol (DAB; 2 mm) or CP-316,819 (CP; 10 µm)) or glycogen removal (amyloglucosidase, 20 U ml-1 ). Tetanic force decreased by (mean (SD)) 21 (15)% (P < 0.001) and 76 (28)% (DAB) or 94 (6)% (CP, P < 0.001) in well-polarized and partially depolarized fibres, respectively. In depolarized fibres, twitch force decreased by 16 (10)% and 55 (26)% with DAB and CP, respectively, with no effect in well-polarized fibres (84 (10)%, P = 0.14). There was no effect of glycogen phosphorylase inhibition on repriming period in well-polarized fibres (median (25th, 75th percentile): 5 (4, 5) vs. 4 (4, 5) ms, P = 0.26), while the repriming period was prolonged from 6 (5, 7) to 8 (7, 10) ms (P = 0.01) in partially depolarized fibres. In line with this, glycogen removal increased repriming period from 5 (5, 6) to 6 (5, 7) ms (P = 0.003) in depolarized fibres. Together, these data strongly indicate that blocking glycogenolysis attenuates Na+ ,K+ -ATPase activity, which in turn increases the repriming period and reduces force, demonstrating a functional link between glycogenolytically derived ATP and force production.

Hypertrophy of human embryonic stem cell-derived cardiomyocytes supported by positive feedback between Ca2+ and diacylglycerol signals.

Human embryonic stem cell-derived cardiomyocytes develop pronounced hypertrophy in response to angiotensin-2, endothelin-1, and a selected mix of three fatty acids. All three of these responses are accompanied by increases in both basal cytoplasmic Ca2+ and diacylglycerol, quantified with the Ca2+ sensor Fluo-4 and a FRET-based diacylglycerol sensor expressed in these cardiomyocytes. The heart glycoside, ouabain (30 nM), and a recently developed inhibitor of diacylglycerol lipases, DO34 (1 µM), cause similar hypertrophy responses, and both responses are accompanied by equivalent increases of basal Ca2+ and diacylglycerol. These results together suggest that basal Ca2+ and diacylglycerol form a positive feedback signaling loop that promotes execution of cardiac growth programs in these human myocytes. Given that basal Ca2+ in myocytes depends strongly on the Na+ gradient, we also tested whether nanomolar ouabain concentrations might stimulate Na+/K+ pumps, as described by others, and thereby prevent hypertrophy. However, stimulatory effects of nanomolar ouabain (1.5 nM) were not verified on Na+/K+ pump currents in stem cell-derived myocytes, nor did nanomolar ouabain block hypertrophy induced by endothelin-1. Thus, low-dose ouabain is not a "protective" intervention under the conditions of these experiments in this human myocyte model. To summarize, the major aim of this study has been to characterize the progression of hypertrophy in human embryonic stem cell-derived cardiac myocytes in dependence on diacylglycerol and Na+ gradient changes, developing a case that positive feedback coupling between these mechanisms plays an important role in the initiation of hypertrophy programs.

Sarcolemmal excitability in the myotonic dystrophies.

INTRODUCTION: Chloride conductance disturbances contribute to sarcolemmal dysfunction in myotonic dystrophy type 1 (DM1) and type 2 (DM2). Studies using muscle velocity recovery cycles (MVRCs) suggest Na+ /K+ -adenosine triphosphatase activation becomes defective in advanced DM1. We used MVRCs to investigate muscle excitability in DM1 and DM2. METHODS: MVRCs were measured for patients with mild (n = 8) and advanced (n = 11) DM1, DM2 (n = 4), and normal controls (n = 30). RESULTS: Residual supernormality after multiple conditioning stimuli was increased in DM2 and advanced DM1. Advanced DM1 was distinguished by increases in muscle relative refractory period (MRRP) and reduced early supernormality as well as peak amplitude decrements for the first and last responses in train during repetitive stimulation. DISCUSSION: Prolongation of the MRRP indicates that depolarization of the resting muscle membrane potential occurs in advanced DM1, with possible implications for future therapeutic approaches. Muscle Nerve 57: 595-602, 2018.

Toxicity of the spiny thick-foot Pachypodium.

PREMISE OF THE STUDY: Pachypodium (Apocynaceae) is a genus of iconic stem-succulent and poisonous plants endemic to Madagascar and southern Africa. We tested hypotheses about the mode of action and macroevolution of toxicity in this group. We further hypothesized that while monarch butterflies are highly resistant to cardenolide toxins (a type of cardiac glycoside) from American Asclepias, they may be negatively affected by Pachypodium defenses, which evolved independently. METHODS: We grew 16 of 21 known Pachypodium spp. and quantified putative cardenolides by HPLC and also by inhibition of animal Na+ /K+ -ATPase (the physiological target of cardiac glycosides) using an in vitro assay. Pachypodium extracts were tested against monarch caterpillars in a feeding bioassay. We also tested four Asclepias spp. and five Pachypodium spp. extracts, contrasting inhibition of the cardenolide-sensitive porcine Na+ /K+ -ATPase to the monarch's resistant form. KEY RESULTS: We found evidence for low cardenolides by HPLC, but substantial toxicity when extracts were assayed on Na+ /K+ -ATPases. Toxicity showed phylogenetic signal, and taller species showed greater toxicity (this was marginal after phylogenetic correction). Application of Pachypodium extracts to milkweed leaves reduced monarch growth, and this was predicted by inhibition of the sensitive Na+ /K+ -ATPase in phylogenetic analyses. Asclepias extracts were 100-fold less potent against the monarch compared to the porcine Na+ /K+ -ATPase, but this difference was absent for Pachypodium extracts. CONCLUSIONS: Pachypodium contains potent toxicity capable of inhibiting sensitive and cardenolide-adapted Na+ /K+ -ATPases. Given the monarch's sensitivity to Pachypodium, we suggest that these plants contain novel cardiac glycosides or other compounds that facilitate toxicity by binding to Na+ /K+ -ATPases.

In vivo assessment of muscle membrane properties in myotonic dystrophy.

INTRODUCTION: Myotonia in myotonic dystrophy types 1 (DM1) and 2 (DM2) is generally attributed to reduced chloride-channel conductance. We used muscle velocity recovery cycles (MVRCs) to investigate muscle membrane properties in DM1 and DM2, using comparisons with myotonia congenita (MC). METHODS: MVRCs and responses to repetitive stimulation were compared between patients with DM1 (n = 18), DM2 (n = 5), MC (n = 18), and normal controls (n = 20). RESULTS: Both DM1 and DM2 showed enhanced late supernormality after multiple conditioning stimuli, indicating delayed repolarization as in MC. Contrary to MC, however, DM1 showed reduced early supernormality after multiple conditioning stimuli, and weak DM1 patients also showed abnormally slow latency recovery after repetitive stimulation. CONCLUSIONS: These findings support the presence of impaired chloride conductance in both DM1 and DM2. The early supernormality changes indicate that sodium currents were reduced in DM1, whereas the weakness-associated slow recovery after repetitive stimulation may provide an indication of reduced Na(+) /K(+) -ATPase activation. Muscle Nerve 54: 249-257, 2016.

Fluid absorption in the isolated midgut of adult female yellow fever mosquitoes (Aedes aegypti).

The transepithelial voltage (Vte) and the volume of isolated posterior midguts of adult female yellow fever mosquitoes (Aedes aegypti) were monitored. In all experiments, the initial Vte after filling the midgut was lumen negative, but subsequently became lumen positive at a rate of approximately 1 mV min(-1). Simultaneously, the midgut volume decreased, indicating spontaneous fluid absorption. When the midguts were filled and bathed with mosquito saline, the average rate of fluid absorption was 36.5±3.0 nl min(-1) (N=4, ±s.e.m.). In the presence of theophylline (10 mmol l(-1)), Vte reached significantly higher lumen-positive values, but the rate of fluid absorption was not affected (N=6). In the presence of NaCN (5 mmol l(-1)), Vte remained close to 0 mV (N=4) and fluid absorption was reduced (14.4±1.3 nl min(-1), N=3, ±s.e.m.). When midguts were filled with buffered NaCl (154 mmol l(-1) plus 1 mmol l(-1) HEPES) and bathed in mosquito saline with theophylline, fluid absorption was augmented (50.0±5.8 nl min(-1), N=12, ±s.e.m.). Concanamycin A (10 µmol l(-1)), ouabain (1 mmol l(-1)), and acetazolamide (1 mmol l(-1)) affected Vte in different ways, but all reduced fluid absorption by 60-70% of the value before addition of the drugs.

A role for the sodium pump in H2O2-induced vasorelaxation in porcine isolated coronary arteries.

Hydrogen peroxide (H2O2) has been proposed to act as a factor for endothelium-derived hyperpolarization (EDH) and EDH may act as a 'back up' system to compensate the loss of the NO pathway. Here, the mechanism of action of H2O2 in porcine isolated coronary arteries (PCAs) was investigated. Distal PCAs were mounted in a wire myograph and pre-contracted with U46619 (1nM-50µM), a thromboxane A2-mimetic or KCl (60mM). Concentration-response curves to H2O2(1µM-1mM), bradykinin (0.01nM-1µM), sodium nitroprusside (SNP) (10nM-10µM), verapamil (1nM-10µM), KCl (0-20mM) or Ca(2+)-reintroduction (1µM-10mM) were constructed in the presence of various inhibitors. Activity of the Na(+)/K(+)-pump was measured through rubidium-uptake using atomic absorption spectrophotometry. H2O2 caused concentration-dependent vasorelaxations with a maximum relaxation (Rmax) of 100±16% (mean±SEM), pEC50=4.18±0.20 (n=4) which were significantly inhibited by PEG-catalase at 0.1-1.0mM H2O2 (P<0.05). 10mM TEA significantly inhibited the relaxation up to 100µM H2O2 (P<0.05). 60mM K(+) and 500nM ouabain significantly inhibited H2O2-induced vasorelaxation producing a relaxation of 40.8±8.5% (n=5) and 47.5±8.6% (n=6) respectively at 1mM H2O2 (P<0.0001). H2O2-induced vasorelaxation was unaffected by the removal of endothelium, inhibition of NO, cyclo-oxygenase, gap junctions, SKCa, IKCa, BKCa Kir, KV, KATP or cGMP. 100µM H2O2 had no effects on the KCl-induced vasorelaxation or Ca(2+)-reintroduction contraction. 1mM H2O2 inhibited both KCl-induced vasorelaxation and rubidium-uptake consistent with inhibition of the Na(+)/K(+)-pump activity. We have shown that the vascular actions of H2O2 are sensitive to ouabain and high concentrations of H2O2 are able to modulate the Na(+)/K(+)-pump. This may contribute towards its vascular actions.

The zebrafish mutant dreammist implicates sodium homeostasis in sleep regulation.

Sleep is a nearly universal feature of animal behaviour, yet many of the molecular, genetic, and neuronal substrates that orchestrate sleep/wake transitions lie undiscovered. Employing a viral insertion sleep screen in larval zebrafish, we identified a novel gene, dreammist (dmist), whose loss results in behavioural hyperactivity and reduced sleep at night. The neuronally expressed dmist gene is conserved across vertebrates and encodes a small single-pass transmembrane protein that is structurally similar to the Na+,K+-ATPase regulator, FXYD1/Phospholemman. Disruption of either fxyd1 or atp1a3a, a Na+,K+-ATPase alpha-3 subunit associated with several heritable movement disorders in humans, led to decreased night-time sleep. Since atpa1a3a and dmist mutants have elevated intracellular Na+ levels and non-additive effects on sleep amount at night, we propose that Dmist-dependent enhancement of Na+ pump function modulates neuronal excitability to maintain normal sleep behaviour.

Temporal dynamics of Na/K pump mediated memory traces: insights from conductance-based models of Drosophila neurons.

Sodium potassium ATPases (Na/K pumps) mediate long-lasting, dynamic cellular memories that can last tens of seconds. The mechanisms controlling the dynamics of this type of cellular memory are not well understood and can be counterintuitive. Here, we use computational modeling to examine how Na/K pumps and the ion concentration dynamics they influence shape cellular excitability. In a Drosophila larval motor neuron model, we incorporate a Na/K pump, a dynamic intracellular Na+ concentration, and a dynamic Na+ reversal potential. We probe neuronal excitability with a variety of stimuli, including step currents, ramp currents, and zap currents, then monitor the sub- and suprathreshold voltage responses on a range of time scales. We find that the interactions of a Na+-dependent pump current with a dynamic Na+ concentration and reversal potential endow the neuron with rich response properties that are absent when the role of the pump is reduced to the maintenance of constant ion concentration gradients. In particular, these dynamic pump-Na+ interactions contribute to spike rate adaptation and result in long-lasting excitability changes after spiking and even after sub-threshold voltage fluctuations on multiple time scales. We further show that modulation of pump properties can profoundly alter a neuron's spontaneous activity and response to stimuli by providing a mechanism for bursting oscillations. Our work has implications for experimental studies and computational modeling of the role of Na/K pumps in neuronal activity, information processing in neural circuits, and the neural control of animal behavior.