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Glutamate levels and lifetime in the brain extracellular space are dinamically regulated by a family of Na+- and K+-dependent glutamate transporters, which thereby control numerous brain functions and play a role in numerous neurological and psychiatric diseases. Migraine is a neurological disorder characterized by recurrent attacks of typically throbbing and unilateral headache and by a global dysfunction in multisensory processing. Familial hemiplegic migraine type 2 (FHM2) is a rare monogenic form of migraine with aura caused by loss-of-function mutations in the α2 Na/K ATPase (α2NKA). In the adult brain, this pump is expressed almost exclusively in astrocytes where it is colocalized with glutamate transporters. Knockin mouse models of FHM2 (FHM2 mice) show a reduced density of glutamate transporters in perisynaptic astrocytic processes (mirroring the reduced expression of α2NKA) and a reduced rate of glutamate clearance at cortical synapses during neuronal activity and sensory stimulation. Here we review the migraine-relevant alterations produced by the astrocytic glutamate transport dysfunction in FHM2 mice and their underlying mechanisms, in particular regarding the enhanced brain susceptibility to cortical spreading depression (the phenomenon that underlies migraine aura and can also initiate the headache mechanisms) and the enhanced algesic response to a migraine trigger. [ABSTRACT FROM AUTHOR]

This study describes the changes in ion homeostasis of human endometrial mesenchymal stem/stromal cells (eMSCs) during the formation of three-dimensional (3D) cell structures (spheroids) and investigates the conditions for apoptosis induction in 3D eMSCs. Detached from the monolayer culture, (2D) eMSCs accumulate Na+ and have dissipated transmembrane ion gradients, while in compact spheroids, eMSCs restore the lower Na+ content and the high K/Na ratio characteristic of functionally active cells. Organized as spheroids, eMSCs are non-proliferating cells with an active Na/K pump and a lower K+ content per g cell protein, which is typical for quiescent cells and a mean lower water content (lower hydration) in 3D eMSCs. Further, eMSCs in spheroids were used to evaluate the role of K+ depletion and cellular signaling context in the induction of apoptosis. In both 2D and 3D eMSCs, treatment with ouabain (1 µM) results in inhibition of pump-mediated K+ uptake and severe K+ depletion as well as disruption of the mitochondrial membrane potential. In 3D eMSCs (but not in 2D eMSCs), ouabain initiates apoptosis via the mitochondrial pathway. It is concluded that, when blocking the Na/K pump, cardiac glycosides prime mitochondria to apoptosis, and whether a cell enters the apoptotic pathway depends on the cell-specific signaling context, which includes the type of apoptotic protein expressed. [ABSTRACT FROM AUTHOR]

This paper describes changes in ionic homeostasis associated with premature senescence in human endometrial mesenchymal stem cells (eMSCs). Changes in the intracellular potassium and sodium content and potassium fluxes through the plasma membrane have been examined/ with flame photometry, It was found that, during oxidative stress-induced cell-cycle arrest and the development of premature aging, eMSCs retained the high ionic heterogeneity that is common for functionally active animal cells. Premature cell aging is accompanied by increased intracellular sodium content and transmembrane potassium fluxes associated with the functioning of the Na/K pump, but does not affect the passive transport of potassium across the plasma membrane. One distinctive feature of stress-induced arrested eMSCs is reduced specific intracellular potassium content (500–600 μmol per 1 g of protein) compared to proliferating eMSCs (800–900 μmol per 1 g of protein). It is suggested that decreased intracellular potassium content associated with the development of premature aging shows the participation of potassium ions in the regulation of cell volume and may indicate a decreased hydration of aging eMSCs. [ABSTRACT FROM AUTHOR]

This study describes the changes in ion homeostasis of human endometrial mesenchymal stem/stromal cells (eMSCs) during the formation of three-dimensional (3D) cell structures (spheroids) and investigates the conditions for apoptosis induction in 3D eMSCs. Detached from the monolayer culture, (2D) eMSCs accumulate Na+ and have dissipated transmembrane ion gradients, while in compact spheroids, eMSCs restore the lower Na+ content and the high K/Na ratio characteristic of functionally active cells. Organized as spheroids, eMSCs are non-proliferating cells with an active Na/K pump and a lower K+ content per g cell protein, which is typical for quiescent cells and a mean lower water content (lower hydration) in 3D eMSCs. Further, eMSCs in spheroids were used to evaluate the role of K+ depletion and cellular signaling context in the induction of apoptosis. In both 2D and 3D eMSCs, treatment with ouabain (1 µM) results in inhibition of pump-mediated K+ uptake and severe K+ depletion as well as disruption of the mitochondrial membrane potential. In 3D eMSCs (but not in 2D eMSCs), ouabain initiates apoptosis via the mitochondrial pathway. It is concluded that, when blocking the Na/K pump, cardiac glycosides prime mitochondria to apoptosis, and whether a cell enters the apoptotic pathway depends on the cell-specific signaling context, which includes the type of apoptotic protein expressed.

Magnesium (Mg 2 +) is an essential mineral for several cellular functions. The concentration of this ion below the physiological concentration induces recurrent neuronal discharges both in slices of the hippocampus and in neuronal cultures. These epileptiform discharges are initially sensitive to the application of N -methyl-D-aspartate (NMDA) receptor antagonists, but these antagonists may lose their effectiveness with prolonged exposure to low [Mg 2 + ], when extracellular Ca 2 + reduction occurs, typical of ictal periods, indicating the absence of synaptic connections. The study herein presented aimed at investigating the effect of reducing the [Mg 2 + ] during the induction of Nonsynaptic Epileptiform Activities (NSEA). As an experimental protocol, NSEA were induced in rat hippocampal dentate gyrus (DG), using a bath solution containing high-K + and zero-added-Ca 2 + . Additionally, computer simulations were performed using a mathematical model that represents electrochemical characteristics of the tissue of the DG granular layer. The experimental results show that the reduction of [Mg 2 + ] causes an increase in the duration of the ictal period and a reduction in the interictal period, intensifying epileptiform discharges. The computer simulations suggest that the reduction of the Mg 2 + level intensifies the epileptiform discharges by a joint effect of reducing the surface charge screening and reducing the activity of the Na/K pump. [ABSTRACT FROM AUTHOR]

Background: In recent times, noncommunicable diseases (NCDs) have attained epidemic status in India and several other countries. Globally, each year, NCDs cause premature deaths of nearly 40 million people younger than 60 years accounting for 70% of all deaths; 80% of these premature deaths are reportedly in the low- and middle-income countries. Many studies have reported increasing prevalence of NCDs such as type 2 diabetes, coronary and pulmonary diseases, and cancer. Incidentally, all these studies besides discussing environmental pollution have grossly ignored dietary profiling of affected populace despite the fact that several studies have established a direct correlation between food quality and good health. Objective: Besides pollutants, an attempt is made to analyze mathematically effects of dietary changes over the last four decades on constantly increasing prevalence of NCDs at cellular level. A relationship seems to exist between increasing prevalence of NCD and large-scale consumption of synthetic salt. Conclusions: Elimination of essential trace elements and micro- and macro-minerals in regular diet through synthetic salt fortified with iodine alone appears to cause more harm than benefits associated with iodization of salt than pollutants. Regular intake of essential trace elements is necessary for normal functioning of many fundamental functions of the body such as Na-K pump, electron–proton transport, biochemistry, and thermodynamics. An attempt has been made to study effects of salt constituents used in food at the most fundamental cellular level by means of disturbances in body electrolyte through heat and ion transport mechanism that is fundamental to various underlying processes in human body. Interestingly, almost all the families who switched over to unprocessed rock salt from synthetic iodized salt reported improvement in general health and reported reduction in medical visits.

The capability of the synchronization modulation (SM) technique in enhancing the function of Na/K pumps has been demonstrated in various cells and tissues, including cardiomyocytes, a monolayer of cultured MDCK kidney cells, peripheral blood vessels, and frog skeletal muscles. This study characterized the membrane potential hyperpolarization induced by SM in both physiological and high [K+]o conditions on single skeletal muscle fibers. The results showed that SM could consistently induce membrane potential hyperpolarization by a few millivolts, and this hyperpolarization was not possible in the presence of ouabain. In contrast, the same electrical pulses but with random frequencies, constant frequencies, or synchronization with backward-modulation could not hyperpolarize the membrane potential. Prolonged field application and higher field intensity enhanced the effects of SM-induced hyperpolarization. Finally, the effect of SM was tested on skeletal muscle fibers incubated in a solution with high external potassium. Results showed that the SM electric field could hyperpolarize the membrane potential even if the external K+ concentration was higher than the normal, which implied the therapeutic effects of the SM electric field on the hyperkalemic situation. [ABSTRACT FROM AUTHOR]

The sodium–potassium pump (Na+/K+ pump) is crucial for cell physiology. Despite great advances in the understanding of this ionic pumping system, its mechanism is not completely understood. We propose the use of a statistical model checker to investigate palytoxin (PTX)-induced Na+/K+ pump channels. We modelled a system of reactions representing transitions between the conformational substates of the channel with parameters, concentrations of the substates and reaction rates extracted from simulations reported in the literature, based on electrophysiological recordings in a whole-cell configuration. The model was implemented using the UPPAAL-SMC platform. Comparing simulations and probabilistic queries from stochastic system semantics with experimental data, it was possible to propose additional reactions to reproduce the single-channel dynamic. The probabilistic analyses and simulations suggest that the PTX-induced Na+/K+ pump channel functions as a diprotomeric complex in which protein–protein interactions increase the affinity of the Na+/K+ pump for PTX.

The Na + /K + -ATPase interacts with the non-selective cation channels TRPC6 but the functional consequences of this association are unknown. Experiments performed with HEK cells over-expressing TRPC6 channels showed that inhibiting the activity of the Na + /K + -ATPase with ouabain reduced the amount of TRPC6 proteins and depressed Ca 2 + entry through TRPC6. This effect, not mimicked by membrane depolarization with KCl, was abolished by sucrose and bafilomycin-A, and was partially sensitive to the intracellular Ca 2 + chelator BAPTA/AM. Biotinylation and subcellular fractionation experiments showed that ouabain caused a multifaceted redistribution of TRPC6 to the plasma membrane and to an endo/lysosomal compartment where they were degraded. The amyloid beta peptide Aβ 1–40 , another inhibitor of the Na + /K + -ATPase, but not the shorter peptide Aβ 1–16 , reduced TRPC6 protein levels and depressed TRPC6-mediated responses. In cortical neurons from embryonic mice, ouabain, veratridine (an opener of voltage-gated Na + channel), and Aβ 1–40 reduced TRPC6-mediated Ca 2 + responses whereas Aβ 1–16 was ineffective. Furthermore, when Aβ 1–40 was co-added together with zinc acetate it could no longer control TRPC6 activity. Altogether, this work shows the existence of a functional coupling between the Na + /K + -ATPase and TRPC6. It also suggests that the abundance, distribution and activity of TRPC6 can be regulated by cardiotonic steroids like ouabain and the naturally occurring peptide Aβ 1–40 which underlines the pathophysiological significance of these processes. [ABSTRACT FROM AUTHOR]

Objective: The mechanisms associated with nerve dysfunction and axonal loss in diabetes has not been fully clarified. Excitability and pathological aspects in nerves from diabetic mice were studied in order to explore the pathophysiology of diabetic neuropathy. Methods: Myelinated nerve fibres from the sciatic nerve of BKS.Cg-m+/+ Leprdb/J mice were studied by registering the CMAP controlled by an automated threshold tracking method. The sciatic nerve was also studied pathologically. Results: Diabetic mice displayed longer latencies, higher thresholds and lower amplitudes compared to controls and had a rightward shift in the stimulus response curves. Strength-duration time constant was lower in diabetic mice but not reaching statistical significance (p = 0.09). Diabetics displayed an increase in accommodation, with a smaller change in excitability in threshold electrotonus. Refractoriness, mean superexcitability and late subexcitability were reduced in diabetic mice. Diabetic mice had a larger number of myelinated fibres compared to controls (p < 0.05), but larger than 9 µm were virtually absent, accounting for near 7 % in control animals. Conclusions: Db/db mice develop electrophysiological changes suggestive of membrane depolarization as the result of Na+ /K+ pump impairment. Loss of large myelinated fibres might also contribute to the nerve excitability profiles in this model. [ABSTRACT FROM AUTHOR]

In rodents, ubiquitous α1-Na, K-ATPase is inhibited by ouabain and other cardiotonic steroids (CTS) at ~10-fold higher concentrations than those effective in other mammals. To examine the specific roles of the CTS- sensitive α1 S- and CTS- resistant α1 R-Na, K-ATPase isoforms, we compared the effects of ouabain on intracellular Na and K content, cell survival, and mitogen-activated protein kinases (MAPK) in human and rat vascular smooth muscle cells (HASMC and RASMC), human and rat endothelial cells (HUVEC and RAEC), and human and rat brain astrocytes. 6-h exposure of HASMC and HUVEC to 3 μM ouabain dramatically increased the intracellular [Na]/[K] ratio to the same extend as in RASMC and RAEC treated with 3000 μM ouabain. In 24, 3 μM ouabain triggered the death of all types of human cells used in this study. Unlike human cells, we did not detect any effect of 3000-5000 μM ouabain on the survival of rat cells, or smooth muscle cells from mouse aorta (MASMC). Unlike in the wild-type α1 mouse, ouabain triggered death of MASMC from α1 mouse expressing human α1-Na, K-ATPase. Furthermore, transfection of HUVEC with rat α1 R-Na, K-ATPase protected them from the ouabain-induced death. In HUVEC, ouabain led to phosphorylation of p38 MAPK, whereas in RAEC it stimulated phosphorylation of ERK1/2. Overall, our results, demonstrate that the drastic differences in cytotoxic action of ouabain on human and rodent cells are caused by unique features of α1S/α1R-Na, K-ATPase, rather than by any downstream CTS-sensitive/resistant components of the cell death machinery. [ABSTRACT FROM AUTHOR]

Intracellular Na ([Na]) is an important modulator of excitation-contraction coupling via regulating Ca efflux/influx, and no investigation has been so far performed in diabetic rat heart. Here, we examined whether any change of [Na] in paced cardiomyocytes could contribute to functional alterations during diabetes. Slowing down in depolarization phase of the action potential, small but significant decrease in its amplitude with a slight depolarized resting membrane potential was traced in live cardiomyocytes from diabetic rat, being parallel with a decreased TTX-sensitive Na channel current ( I) density. We recorded either [Na] or [Ca] by using a fluorescent Na indicator (SBFI-AM or Na-Green) or a Ca indicator (Fura 2-AM) in freshly isolated cardiomyocytes. We examined both [Na] and [Ca] at rest, and also [Na] during pacing with electrical field stimulation in a range of 0.2-2.0 Hz stimulation frequency. In order to test the possible contribution of Na/H exchanger (NHE) to [Na], we examined the free cytoplasmic [H] changes from time course of [H] recovery in cardiomyocytes loaded with SNARF1-AM by using ammonium prepulse method. Our data showed that [Na] in resting cells from either diabetic or control group was not significantly different, whereas the increase in [Na] was significantly smaller in paced diabetic cardiomyocytes compared to that of the controls. However, resting [Ca] in diabetic cardiomyocytes was significantly higher than that of the controls. Here, a lower basal pH in diabetics compared with the controls correlates also with a slightly higher but not significantly different NHE activity and consequently a similar Na loading rate at resting state with a leftward shift in pH sensitivity of NHE-dependent H-flux. NHE protein level remained unchanged, while protein levels of Na/K ATPase and Na/Ca exchanger were decreased in the diabetic cardiomyocytes. Taken together, the present data indicate that depressed I plays an important role in altered electrical activity with less Na influx during contraction, and an increased [Ca] load in these cells seems to be independent of [Na]. The data with insulin treatment suggest further a recent balance between Na influx and efflux proteins associated with the [Na], particularly during diabetes. [ABSTRACT FROM AUTHOR]

In animal cells, the resting potential is established by the concentration gradients of sodium and potassium ions and the different permeabilities of the cell membrane to them. The large concentration gradients of sodium and potassium ions are maintained by the Na/K pump. Under physiological conditions, the pump transports three sodium ions out of and two potassium ions into the cell per ATP hydrolyzed. However, unlike other primary or secondary active transporters, the Na/K pump does not work at the equilibrium state, so the pumping ratio is not a thermodynamic property of the pump. In this article, I propose a dipole-charging model of the Na/K pump to prove that the three Na to two K pumping ratio of the Na/K pump is determined by the ratio of the ionic mobilities of potassium to sodium ions, which is to ensure the time constant τ and the τ-dependent processes, such as the normal working state of the Na/K pump and the propagation of an action potential. Further, the concentration ratios of potassium ions outside and inside the cell to sodium ions inside and outside the cell are 0.3027 and 0.9788, respectively, and the sum of the potassium and sodium equilibrium potentials is −30.3 mV. A comparative study on these constants is made for some marine, freshwater and terrestrial animals. These findings suggest that the pumping ratio of the Na/K pump and the ion concentration ratios play a role in the evolution of animal cells. [ABSTRACT FROM AUTHOR]

Hyperglycaemia is well known to cause reductions in plasma Na levels or even hyponatraemia due to an osmotically induced dilution of the interstitium and blood. It is, however, unclear whether this dilution is significantly counteracted by ion regulatory homeostatic mechanism(s) or not. Furthermore, the effects of moderate hyperglycaemia on other major ions are less well known. To further clarify these questions, we measured the changes in blood osmolarity and concentrations of Na, K, Cl, Mg and Ca during a 4-h-long experimental hyperglycaemia in healthy subjects rendered temporarily insulin deficient using the hyperglycaemic clamp. Hyperglycaemia, 16.8 mM, was rapidly imposed from a baseline of 4.4 mM by intravenous somatostatin and glucose infusions in 19 healthy subjects (10 m, 9 f; age 36 ± 5 years (mean ± SD); BMI 22.7 ± 2.9 kg/m²). Subsequently, glycaemia was returned to basal and measurements continued until all dynamic changes had stopped (at ~8 h). Osmolarity increased from 281.8 ± 0.7 to 287.9 ± 0.7, while Na decreased from 143.9 ± 0.3 to 138.7 ± 0.2, Cl from 101.7 ± 0.2 to 99.5 ± 0.1, Ca from 1.98 ± 0.04 to 1.89 ± 0.02 and Mg from 0.84 ± 0.01 to 0.80 ± 0.00 mM. All these changes were rapidly reaching stable levels. K increased from 4.02 ± 0.02 to 4.59 ± 0.02 mM ( P < 0.0001) also reaching stable levels but with some delay. Na, Cl, Mg and Ca are essentially determined by blood dilution, and their values will remain diminished as long as the hyperglycaemia lasts. Partial suppression of insulin-stimulated Na/K pumping lead to increased K levels. The combination of elevated K and decreased Mg and Ca levels may lead to an altered excitability, which is particularly relevant for diabetic patients with heart disease. [ABSTRACT FROM AUTHOR]

Na/K pump activity and metabolic rate are both higher during the day in the suprachiasmatic nucleus (SCN) that houses the circadian clock. Here we investigated the role of intracellular Na+ and energy metabolism in regulating Na/K pump activity and neuronal excitability. Removal of extracellular K+ to block the Na/K pump excited SCN neurons to fire at higher rates and return to normal K+ to reactivate the pump produced rebound hyperpolarization to inhibit firing. In the presence of tetrodotoxin to block the action potentials, both zero K+ -induced depolarization and rebound hyperpolarization were blocked by the cardiac glycoside strophanthidin. Ratiometric Na+ imaging with a Na+ -sensitive fluorescent dye indicated saturating accumulation of intracellular Na+ in response to pump blocKade with zero K+. The Na+ ionophore monensin also induced Na+ loading and hyperpolarized the membrane potential, with the hyperpolarizing effect of monensin abolished in zero Na+ or by pump blockade. Conversely, Na+ depletion with Na+ -free pipette solution depolarized membrane potential but retained residual Na/K pump activity. Cyanide inhibition of oxidative phosphorylation blocked the Na/K pump to depolarize resting potential and increase spontaneous firing in most cells, and to raise intracellular Na+ levels in all cells. Nonetheless, the Na/K pump was incompletely blocked by cyanide but completely blocked by iodoacetate to inhibit glycolysis, indicating the involvement of both oxidative phosphorylation and glycolysis in fueling the Na/K pump. Together, the results indicate the importance of intracellular Na+ and energy metabolism in regulating Na/K pump activity as well as neuronal excitability in the SCN neurons. [ABSTRACT FROM AUTHOR]

The spontaneously active Br neuron from the brain-subesophageal ganglion complex of the garden snail Helix pomatia rhythmically generates regular bursts of action potentials with quiescent intervals accompanied by slow oscillations of membrane potential. We examined the involvement of the Na/K pump in modulating its bursting activity by applying a static magnetic field. Whole snail brains and Br neuron were exposed to the 10-mT static magnetic field for 15 min. Biochemical data showed that Na/K-ATPase activity increased almost twofold after exposure of snail brains to the static magnetic field. Similarly, P NMR data revealed a trend of increasing ATP consumption and increase in intracellular pH mediated by the Na/H exchanger in snail brains exposed to the static magnetic field. Importantly, current clamp recordings from the Br neuron confirmed the increase in activity of the Na/K pump after exposure to the static magnetic field, as the magnitude of ouabain's effect measured on the membrane resting potential, action potential, and interspike interval duration was higher in neurons exposed to the magnetic field. Metabolic pathways through which the magnetic field influenced the Na/K pump could involve phosphorylation and dephosphorylation, as blocking these processes abolished the effect of the static magnetic field. [ABSTRACT FROM AUTHOR]

Blood vessel dilation starts from activation of the Na/K pumps and inward rectifier K channels in the vessel smooth muscle cells, which hyperpolarizes the cell membrane potential and closes the Ca channels. As a result, the intracellular Ca concentration reduces, and the smooth muscle cells relax and the blood vessel dilates. Activation of the Na/K pumps and the membrane potential hyperpolarization plays a critical role in blood vessel functions. Previously, we developed a new technique, synchronization modulation, to control the pump functions by electrically entraining the pump molecules. We have applied the synchronization modulation electric field noninvasively to various intact cells and demonstrated the field-induced membrane potential hyperpolarization. We further applied the electric field to blood vessels and investigated the field induced functional changes of the vessels. In this paper, we report the results in a study of the membrane potential change in the smooth muscle cells of mesenteric blood vessels in response to the oscillating electric field. We found that the synchronization modulation electric field can effectively hyperpolarize the muscle membrane potential quickly in seconds under physiological conditions. [ABSTRACT FROM AUTHOR]

At present, when the technological progress brings progressive increase in environmental pollutions by different chemical and physical (ionizing and non-ionizing radiations) factors, the detection of the safety of environmental medium from the point of public health is one of the fundamental problems of modern Life Sciences. This problem has especially disquieting character after the Chernobyl and Japan nuclear catastrophes, when the level of background ionizing radiation and chemical pollutions of environmental medium of the number of world's regions are increased beyond safety doses. As the biological effect of weak environmental factors have nonlinear dose-dependent character, besides its thermodynamic characteristics it depends also on environmental composition and initial state of organism. Therefore, the current policy of World Health Organization and other international organizations whose mission is to establish safety standards for environmental pollutions by chemical and physical factors, based only on the their concentration or energy absorption rate by organism cannot be considered as adequate. It is suggested that the biological marker having universal sensitivity to different factors and determining the functional state of organisms could be used for estimation of the safety doses of environmental factors on organism. In present review are presented the data consisting of the hypothesis according to which the Na/K pump and Na/Ca-controlling cell hydration could serve as a universal and extra-sensitive cellular marker for detection of hazardous effect of environmental pollutions. [ABSTRACT FROM AUTHOR]

It was shown that the 4 Hz 30 dB mechanical vibration (MV) of physiological solution (PS) had modulation effect on snail heart contractility. However, the nature of metabolic pathway of MV-treated PS-induced effect is not clear yet. It was suggested that the MV-induced modulation of water molecules dissociation leads to the variation of reactive oxygen species' level in cell bathing medium, which could serve as a messenger for switching on the metabolic pathway(s) responsible for modulation of muscle contractility. The aim of present work was to check this hypothesis and to elucidate the metabolic pathway through which the effect of MV-treated PS on heart contractility was realized. For this purpose, the effect of MV on heat fusion periods (HFP) and HO content in PS, as well as on heart contractility, Ca efflux, intracellular levels of cGMP and cAMP, muscle hydration, and ouabain binding were studied. It was shown that MV treatment of PS increased the HFP-(21.33 ± 4%) and decreased the HO content-(5 ± 0.9%). The intracardiac perfusion by MV-treated PS increased the amplitudes of heart contractility, which was accompanied by the increasing of Ca efflux (252.4 ± 16%), elevation of cGMP's level (42.05 ± 7%), decreasing of cAMP's level (82.67 ± 7%), increasing of the tissue hydration (18.64 ± 3%), and increasing of the number of ouabain binding sides (25 ± 4%). It was suggested that MV-induced increasing of heart muscle contraction amplitudes is due to the decreases of HO content in the medium, which leads to the elevation of heart muscle contractility in result of activation of cGMP-dependent Na/Ca exchange in forward regime. [ABSTRACT FROM AUTHOR]

Although the high sensitivity of the Na/K pump in cell membrane to ionizing radiation is well known in literature, the individual role of different isoforms of pump in determination of its radio-sensitivity is not clear yet. This is the subject of the present investigation. Using isotope, electro-physiological and enzymological methods, the effect of γ-ionizing radiation on cell membrane voltage-current characteristics, acetylcholine-induced membrane current, Na and Ca exchange between cells and bathing solution, Na+K-ATPase activity, dose-dependent ouabain binding with cell membrane, intracellular cAMP and membrane phosphorylation in snail neurons were studied. The changes in neurons as a result of 30-min γ-radiation exposure of snails to 5.16 Ci/kg at the end of the first 30 min of post-radiation period were as follows: the increase in membrane ionic conductance reversed the ouabain sensitivity of acetylcholine-induced currents, stimulation of Na and Ca uptakes, inhibition of Na/K pump, activation Na/Ca exchange in reversed mode, increase in ouabain binding with high-affinity α and decrease with α middle-affinity receptors, decrease in intracellular cAMP content and membrane dephosphorylation. On the basis of the obtained data, it is suggested that both α and α catalytic subunits of Na+K-ATPase serve primary membrane sensors through the activation of which the biological effect of γ-radiation on neurons is realized. The IR has activation effects on α-dependent Na/Ca exchange in forward and its inactivation on α-dependent reverse modes. [ABSTRACT FROM AUTHOR]

Our previous studies have shown that a specially designed, so-called synchronization modulation electric field can entrain active transporter Na/K pumps in the cell membrane. This approach was previously developed in a study of single cells using a voltage clamp to monitor the pump currents. We are now expanding our study from isolated single cells to aggregated cells in a 3-dimensional cell matrix, through the use of a tissue slice from the rat heart. The slice is about 150 μm in thickness, meaning the slices contain many cell layers, resulting in a simplified 3-dimensional system. A fluorescent probe was used to identify the membrane potential and the ionic concentration gradients across the cell membrane. In spite of intrinsic cell-to-cell interactions and the difficulty in stimulating cell aggregation in the tissue slice, the oscillating electric field increased the intracellular fluorescent intensity, indicating elevation of the cell ionic concentration and hyperpolarization of the cell membrane. Blockage of these changes by ouabain confirmed that the results are directly related to Na/K pumps. These results along with the backward modulation indicate that the synchronization modulation electric field can influence the Na/K pumps in tissue cells of a 3-dimensional matrix and therefore hyperpolarize the cell membrane. [ABSTRACT FROM AUTHOR]

The calcium ion is one of the most versatile, ancient, and universal of biological signaling molecules, known to regulate physiological systems at every level from membrane potential and ion transporters to kinases and transcription factors. Disruptions of intracellular calcium homeostasis underlie a host of emerging diseases, the calciumopathies. Cytosolic calcium signals originate either as extracellular calcium enters through plasma membrane ion channels or from the release of an intracellular store in the endoplasmic reticulum (ER) via inositol triphosphate receptor and ryanodine receptor channels. Therefore, to a large extent, calciumopathies represent a subset of the channelopathies, but include regulatory pathways and the mitochondria, the major intracellular calcium repository that dynamically participates with the ER stores in calcium signaling, thereby integrating cellular energy metabolism into these pathways, a process of emerging importance in the analysis of the neurodegenerative and neuropsychiatric diseases. Many of the calciumopathies are common complex polygenic diseases, but leads to their understanding come most prominently from rare monogenic channelopathy paradigms. Monogenic forms of common neuronal disease phenotypes—such as seizures, ataxia, and migraine—produce a constitutionally hyperexcitable tissue that is susceptible to periodic decompensations. The gene families and genetic lesions underlying familial hemiplegic migraine, FHM1/CACNA1A, FHM2/ATP1A2, and FHM3/SCN1A, and monogenic mitochondrial migraine syndromes, provide a robust platform from which genes, such as CACNA1C, which encodes the calcium channel mutated in Timothy syndrome, can be evaluated for their role in autism and bipolar disease. [ABSTRACT FROM AUTHOR]

Na,K-ATPase is a crucial enzyme for ion homeostasis in human tissues. Different isozymes are produced by assembly of four α- and three β-subunits. The expression of the α3/β1 isozyme is confined to brain and heart. Its heterologous production has so far never been attempted in a lower eukaryote. In this work we explored whether the methylotrophic yeast Pichia pastoris is capable of expressing the α3/β1 isoform of human Na,K-ATPase. cDNAs encoding the α3 and the β1-subunits were cloned under the control of the inducible promoter of Pichia pastoris alcohol oxidase 1. Pichia pastoris could express the single α3- and β1-subunits and even coexpress them after methanol induction. β1-subunit was produced as a major 44-kDa glycosylated polypeptide and α3 as a 110-kDa unglycosylated polypeptide. Expression at the plasma membrane was limited in shaking flask cultures but by cultivating P. pastoris cells in a fermenter there was a 10-fold increase of the number of ouabain binding sites per cell. The exported enzyme was estimated to be about 0.230 mg L−1 at the end of a bioreactor run. Na,K-ATPase proved active and the dissociation constant of the recombinant enzyme-ouabain interaction was determined. [ABSTRACT FROM AUTHOR]

FXYD1 is a transmembrane protein predominantly expressed in excitable tissues that associates with and regulates Na/K ATPase. PKA phosphorylates FXYD1 at serine 68 (S68), however, the effects of phosphorylation on Na/K ATPase activity are not fully characterized. The objectives of this study were to characterize Na/K ATPase currents in FXYD1 wild-type (WT) and knockout (KO) adult mouse ventricular myocytes, and investigate the effects of FXYD1 on Na/K ATPase currents using the whole-cell patch-clamp technique. A peptide representing the 19 C-terminal residues of FXYD1 (FXYD154-72) was introduced into the interior of FXYD1 KO and WT myocytes through the patch pipette. K-sensitive Na/K ATPase currents were higher in KO myocytes (2.9±0.1 pA/pF; n=4) compared with WT (1.9±0.1 pA/pF; n=4). Unphosphorylated FXYD154-72, at a concentration of 4 µM, reduced the currents in WT (from 2.1±0.1 to 1.3±0.1 pA/pF; P<0.05, n=7) and KO (from 2.9±0.1 to 1.7±0.1 pA/pF; P<0.05, n=5), whereas, 1 µM of FXYD154-72 phosphorylated at S68 increased currents in WT (from 1.91±0.09 to 3.1±0.5 pA/pF; P<0.05, n=6) and KO (from 2.7±0.11 to 3.8±0.2 pA/pF; P<0.05, n=6) myocytes. Coimmunoprecipitation studies demonstrated that S68 phosphorylated and unphosphorylated FXYD154-72 associates with Na/K ATPase α1 subunit. We conclude that unphosphorylated FXYD1 inhibits Na/K ATPase, whereas S68 phosphorylated FXYD1 stimulates Na/K ATPase to a level above that seen in the absence of FXYD1. [ABSTRACT FROM AUTHOR]

Increasing evidence indicates that modulation of Na+/K+ ATPase activity is involved in forms of neuronal and synaptic plasticity. In tactile (T) neurons of the leech Hirudo medicinalis, Na+/K+ ATPase is the main determinant of the afterhyperpolarization (AHP), which characterizes the firing of these mechanosensory neurons. Previously, it has been reported that cAMP (3′,5′-cyclic adenosine monophosphate), which mediates the effects of serotonin (5HT) in some forms of learning in the leech, negatively modulates Na+/K+ ATPase activity, thereby reducing the AHP amplitude in T neurons. Here, we show that a transient inhibition of Na+/K+ ATPase can affect the synaptic connection between two ipsilateral T neurons. Bath application of 10 nm dihydroouabain (DHO), an ouabain analogue, causes an increase in the amplitude of the synaptic potential (SP) recorded in the postsynaptic element when a test stimulus is applied in the presynaptic neuron. Iontophoretic injection of cAMP into the presynaptic T neuron also produces an increase of SP. Simulations carried out by using a computational model of the T neuron suggest that a reduction of the pump rate and a consequent depression of the AHP might facilitate the conduction of action potentials to the synaptic terminals. Moreover, nearly intact leeches injected with 10 nm DHO respond with a swimming episode more quickly to an electrical stimulation, which selectively activates T neurons exhibiting sensitization of swimming induction. Collectively, our results show that inhibition of Na+/K+ ATPase is critical for short-term plasticity. [ABSTRACT FROM AUTHOR]

Abstract: There has been increasing evidence that tumor necrosis factor alpha (TNF-α) is synthesized by cardiomyoctes and contributes to their impaired function and to cardiac failure. Because the Na+–K+ ATPase is a key player in the contraction of cardiomyocytes, this work was undertaken to study the effect of TNF-α on the Na+–K+ ATPase in rat heart. Sprague Dawley rats (Rattus norvegicus) were injected with TNF-α (270 ng/100 g body weight) and 4 h later the ventricles were isolated, homogenized and assayed for their Na+–K+ ATPase activity. The effect of TNF-α on the pump was studied also in isolated myocytes treated in suspension. The involvement of PGE2 was investigated by pre-treating animals or cells with indomethacin, an inhibitor of COX enzymes. The involvement of NF-κB and AP-1 was studied using their respective inhibitors PDTC and curcumin. A time response study showed an increase in the activity of the Na+–K+ ATPase in the left and right ventricles of animals treated with the cytokine, with no change in its protein expression. This effect disappeared in the presence of indomethacin suggesting an involvement of PGE2 in the action of TNF-α. Rats and cells treated directly with PGE2 showed a dose-dependent response. A decrease in the activity of the Na+–K+ ATPase was observed at a low dose and an increase at a high dose in both ventricles. Since PGE2 is suspected to be the active mediator in TNF-α signaling, inhibiting its synthesis by inhibiting some suspected transcription factors was attempted. PDTC abrogated fully, and curcumin partially the effect of the cytokine. It was concluded that TNF-α activates NF-κB and AP-1 and induces PGE2 release which alters dose-dependently the activity of the pump by activating different EP receptors with different affinities for PGE2. [Copyright &y& Elsevier]

We experimentally studied the Na/K pump currents evoked by a train of squared pulses whose pulse-duration is about the time course of Na-extrusion at physiological conditions. The magnitude of the measured pump current can be as much as three-fold of that induced by the traditional single pulse measurement. The increase in the pump current is directly dependent on the number of pre-pulses. The larger the number of the pre-pulses is, the higher the current magnitude can be obtained. At a particular number of pre-pulses, the pump current becomes saturated. These results suggest that a large number of pre-pulses may synchronize the pump molecules to work at the same pace. As a result, the pump molecules may extrude Na ions at the same time corresponding to the stimulation pulses, and pump in K ions at the same time during the pulse intervals. Therefore, the measured pump current is three-fold of that measured by a single pulse where the outward and inward pump currents are canceled each other. [ABSTRACT FROM AUTHOR]

Abstract: The major K influx pathways and their response to thiol modification by N-ethylmaleimide (NEM) and protein kinase and phosphatase inhibitors were characterized in human lens epithelial B3 (HLE-B3) cells with Rb as K congener. Ouabain (0.1mM) and bumetanide (5μM) discriminated between the Na/K pump (∼35% of total Rb influx) and Na–K–2Cl cotransport (NKCC) (∼50%). Cl-replacement with nitrate or sulfamate revealed <10% residual [ouabain+bumetanide]-insensitive K–Cl cotransport (KCC). At 0.3–0.5mM, NEM stimulated the Na/K pump by 2-fold independent of external Na, KCC between 2 and 4-fold, and abolished ∼90% of NKCC. Calyculin-A, a serine/threonine protein phosphatase-1 inhibitor, did not affect NKCC but inhibited KCC, whereas 10μM staurosporine, a serine/threonine kinase inhibitor, abolished NKCC, and stimulated KCC only when followed by NEM treatment. The tyrosine-kinase inhibitor genistein, at concentrations >100μM, activated the Na/K pump and abolished NKCC but did not affect KCC. The data suggest at least partial inverse regulation of KCC and NKCC in HLE-B3 cells by signaling cascades involving serine, threonine and tyrosine phosphorylation/dephosphorylation equilibria. [Copyright &y& Elsevier]

Abstract: The present study provides the first evidence that the abundance of catalytic α1-subunit of Na,K-ATPase increases in the course of T cell blast transformation. Immunodepressant cyclosporin A at anti-proliferative doses diminished the induction of α1 protein in activated lymphocytes. Furthermore, in competent T cells, IL-2 increases both the transport activity of Na/K pump and the content of Na,K-ATPase α1 protein in a time-dependent manner. A correlation was found between the long-term elevation in ouabain-sensitive Rb influxes and the increase in α1 protein content in late activated T cells. These results suggest that (1) the increased expression of Na,K-ATPase proteins underlie the cell cycle-dependent upregulation of ion pump during T cell transformation, and (2) IL-2 is involved in the regulated expression of Na,K-ATPase in human lymphocytes. [Copyright &y& Elsevier]

We outline the basic operational, structural and functional features of ion motive ATPases: trans-membrane proteins central to biological functions of all animal cells. As an example we discuss the modeling problems associated with the operation of the surface membrane Na+,K+-ATPase and skeletal muscle sarcoplasmic reticulum Ca2+-ATPase and focus on the frameworks required for their solution. There are three basic problems: identification of the pathway for ion permeation, prediction of ion binding rate coefficients and affinities based on the structure of the protein, and prediction of conformational changes of protein structure and the associated movement of charges within the membrane dielectric. A solution strategy useful in approaching the first two problems and preliminary results obtained using molecular dynamics simulations are also presented. [ABSTRACT FROM AUTHOR]

This study concerns the modulatory effects of the gamma modulator of the Na/K pump, in particular whether the effects seen in previous experiments with isolated membranes are relevant to Na/K pump behavior in intact mammalian cells. For this purpose, HeLa cells previously transfected with the rat Na/K catalytic subunit were used. The results show that both variants of the regulator, gammaa and gammab, decrease the apparent affinity of the pump for Na(+) and cause a modest increase in apparent ATP affinity as seen in measurements of ouabain-sensitive (86)Rb(K(+)) influx into cells in which ATP was varied using antimycin A and glucose. Equivalent results had been obtained previously in our analyses of Na,K-ATPase activity of membrane fragments, i.e., an increase in K(0.5(Na)) at high K(+) concentration and a decrease in K'(ATP). Comparison of clones of gamma-transfected and mock-transfected cells (with similar V(max) values) indicated that gamma causes a modest approximately 30% increase in the steady-state concentration of intracellular Na(+). Furthermore, for both gammaa and gammab, values of intracellular Na(+) were similar to those predicted from the kinetic constants, K(0.5(Na)) and V(max). Finally, there was a gamma-mediated increase in apparent affinity for extracellular K(+), which had not been detected in assays of permeabilized membranes. [ABSTRACT FROM AUTHOR]

Shrinkage is the earliest hallmark of cells undergoing apoptosis. This study examines the role of this phenomenon in the onset of vascular smooth muscle cell (VSMC) apoptosis triggered by growth factor withdrawal. In hyperosmotic media, VSMC showed the same amplitude of shrinkage but were more resistant to apoptosis than endothelial, epithelial and immune system cells. As with growth factor withdrawal, apoptosis in hyperosmotically-shrunken VSMC was sharply potentiated by transfection with E1A-adenoviral protein and was suppressed by activation of cAMP signaling as well as by the pan-caspase inhibitor z-VAD.fmk. Both cell shrinkage and apoptosis in VSMC-E1A treated with hyperosmotic medium were potentiated under sustained Na+, K+ pump inhibition with ouabain that was in contrast to inhibition of apoptosis documented in ouabain-treated, serum-deprived cells. After 1-hr incubation in serum-deprived medium, VSMC-E1A volume declined by ∼15%. Transfer from hypotonic to control medium decreased VSMC-E1A volume by ∼25% without any induction of apoptosis. Neither swelling in hyposmotic medium nor dissipation of the transmembrane gradient of K+ and major organic osmolytes protected serum-deprived VSMC-E1A from apoptosis. Thus, our results show that similarly to immune system, endothelial and epithelial cells, extensive VSMC shrinkage in hyperosmotic medium leads to the development of apoptosis. In contrast to hyperosmotic medium, the modest cell volume decrease occurring in serum-deprived VSMC does not contribute to triggering of the apoptotic machinery. [ABSTRACT FROM AUTHOR]

The Na/K pump plays a key role in the regulation of the intracellular concentrations of monovalent cations and related cell function leading to electrogenesis and excitation–contraction coupling. We focus this review on the analysis of recent data showing that (i) inhibition of the Na/K pump triggers a signaling cascade independently of modulation of the intracellular [Na+]i/[K+]i ratio; (ii) elevation of [Na+]i under sustained inhibition of the Na/K pump leads to expression of a set of genes by [Ca2+]i-dependent and independent pathways; (iii) [Na+]i-sensitive genes are involved in the inhibition of programmed cell death (apoptosis) in vascular smooth muscle cells. [ABSTRACT FROM AUTHOR]

The Na/K pump of vascular smooth muscle cells (VSMC) and renal epithelial cells (REC) is viewed as a target of digitalis and endogenous ouabain (EO), leading to the development of hypertension. In this study, we compared the effect of ouabain on Na/K pump activity and the intracellular content of monovalent cations in VSMC and REC obtained from rats, humans and dogs. In VSMC from the rat aorta, ouabain inhibited maximal Na/K pump activity measured as the rate of [sup 86]Rb influx in Na[sup +]-loaded cells, with an ID[sub 50] of ~20-30 µM without any differences between two strains of normotensive rats (WKY and BN.lx) and three substrains of spontaneously hypertensive rats (SHR). Half-maximal inhibition of the Na/K pump in REC from the rat inner medullary collecting duct was observed at ~20 µM of ouabain. In contrast to rat cells, half-maximal inhibition of [sup 86]Rb influx in VSMC from human coronary arteries and in REC from the Madin-Darby canine kidney was seen at ~0.03 and 0.1 µM ouabain, respectively. At concentrations lower than 100 µM, ouabain did not affect the intracellular content of exchangeable Na[sup +] and K[sup +] in rat VSMC, measured as the steady-state distribution of [sup 22]Na and [sup 86]Rb, whereas in human VSMC, it increased the intracellular Na[sup +]/K[sup +] ratio with an ID[sub 50] of ~0.5 µM. Keeping in mind that the circulating level of administered digitalis and EO does not exceed 10[sup -9] M, our results strongly suggest that the involvement of these compounds in the pathogenesis of hypertension in rats is not mediated by inhibition of the α1-isoform of the Na/K pump in VSMC and REC. Alternative mechanisms of the involvement of EO and ouabain-like factors in the development of hypertension are considered. [ABSTRACT FROM AUTHOR]

The influence of inhibiting the inward rectifier and Na/K pump on endothelium-dependent hyperpolarizations in smooth muscle cells of the mesenteric artery was investigated. Membrane potential was measured with microelectrodes, and the influence of low concentrations of Ba2+ (30 µM) and of high concentrations of ouabain (0.5 mM) on smooth muscle hyperpolarization elicited by prolonged or by transient exposure to acetylcholine (ACh, 3×10–7 M) was assessed in the continuous presence of NG-nitro-l-arginine (100 µM) and indomethacin (50 µM). Pre-exposure to Ba2+ did not inhibit the magnitude of smooth muscle cell hyperpolarization induced by ACh superfusion, but significantly slowed its onset and time course. The membrane potential response to transient ACh applications, however, was impaired. After combined Ba2+ and ouabain pre-exposure, peak hyperpolarizations to ACh superfusion were somewhat decreased but not abolished. In addition, 4–5 mM increases of the extracellular K+ concentration consistently depolarized smooth muscle cells. These findings argue against the idea that smooth muscle inward rectifier K+ channels and Na/K pumping play a role in the ACh-induced endothelium-dependent hyperpolarization of this preparation. Moreover, the slowing of smooth muscle membrane hyperpolarization by Ba2+ is discussed in terms of the influence of this ion on the release of hyperpolarizing factor. [ABSTRACT FROM AUTHOR]

The β-agonist isoproterenol (ISO) reduces the Na/K pump current ( Ip) via β-adrenergic receptors when the intracellular calcium concentration ([Ca2+]i) is below 150 nM [8]. In the present study, the intracellular signaling pathway was investigated with whole-cell patch-clamp of isolated guinea pig ventricular myocytes. The inhibitory effect of ISO could be mimicked by external application of the membrane-permeant cAMP analog chlorophenylthio-cAMP (0.5 mM), the phosphodiesterase inhibitor isobutyl-1-methylxanthine (IBMX, 100 μM), or the adenylyl cyclase activator forskolin (50 μM). Intracellular application of the synthetic peptide inhibitor of protein kinase A (PKA), PKI (5 μM), prevented the effect of ISO. These results suggest that the inhibitory effect of ISO on Ip is mediated via a phosphorylation step induced by a cAMP-dependent PKA pathway. Neither the non-specific protein kinase inhibitor H7 (100 μM) nor the protein phosphatase inhibitor calyculin A (0.5 μM) had any effect on Ip in the absence of ISO. However, H7 could increase Ip and calyculin A could reduce it in the presence of ISO (1 μM and 12 nM respectively). These results indicate that there is a low basal level of phosphorylation which makes the effects of H7 and calyculin A difficult to detect in the absence of an ISO-induced increase in phosphorylation level. [ABSTRACT FROM AUTHOR]

Isolated microperfused rabbit renal proximal tubule S2 segments, if incubated in conventional substrate containing HCO3 –Ringer solution, exhibit lower cell membrane potentials ( Vb) and elevated intracellular Na+ concentrations ([Na]i) compared to rat tubules in vivo. Assuming that these and other differences reflect insufficient metabolic and/or hormonal stimulation of the cells, we have used microelectrode techniques to test whether improving substrate supply and applying norepinephrine (NE, to compensate for the missing nerve supply) reverts Vb and [Na]i to values observed in vivo. Application of D-glucose (5.5 mmol/l) and additional application of pyruvate, lactate, or L-alanine (each 10 mmol/l), or bathing the tubules in Dulbecco’s modified Eagle’s tissue culture medium (DMEM) significantly increased Vb as compared to substrate-free or D-glucose-containing control solution and these effects could be prevented – as tested in the case of pyruvate – by inhibition of the Na/K pump with ouabain. However, high concentrations of acetate, β-hydroxybutyrate, or L-glutamine had no significant effect. The largest effect was obtained with joint application of DMEM and NE (10 μmol/l) which increased Vi as compared to substrate-free or D-glucose-containing control solution and these effects could be prevented – as tested in the case of pyruvate – by inhibition of the Na/K pump with ouabain. However, high concentrations of acetate, β-hydroxybutyrate, or L-glutamine had no significant effect. The largest effect was obtained with joint application of DMEM and NE (10 μmol/l) which increased Vb from –42.8 ± 1.3 mV (SEM) to –55.3 ± 2.5 mV ( n = 11). Interestingly we noticed that under the latter conditions the Vb response to bath application of 1 mmol/l amiloride virtually disappeared, i.e. it changed from a depolarization of +14.6 ± 1.4 mV (in D-glucose Ringer solution) to +0.6 ± 0.7 mV (in DMEM plus NE) ( n = 8), with some tubules showing even a small hyperpolarization. The latter implies partial restoration of the in vivo behaviour, since in experiments on rat proximal tubules in vivo amiloride regularly hyperpolarized the cells (by –3.4 ± 0.76 mV, n = 5). Obviously under conventional in vitro conditions an amiloride-inhibitable K+ conductance is activated which is inactive in vivo and also inactivates under improved conditions in vitro. In agreement with observations reported in the subsequent publication our results demonstrate that isolated proximal tubules undergo functional alterations which may be largely prevented by improved metabolic and stimulatory incubation conditions. [ABSTRACT FROM AUTHOR]

The outer hair cell isolated from the guinea-pig was superfused in vitro and the cytosolic calcium concentration ([Ca]) and sodium concentration ([Na]) were measured using fluorescence indicators. Under the resting condition, [Ca] and [Na] were 91±9 nM ( n = 51) and 110±5 mM ( n = 12), respectively. Removal of external Na by replacing with N-methyl-D-glucamine (NMDG) increased [Ca] by 270±79% ( n = 27) and decreased [Na] by 23±4 mM ( n = 6). Both changes in [Ca] and [Na] were totally reversible on returning external Na to the initial value and were inhibited by addition of 0.1 mM La or 100 μM amiloride 5-( N,N-dimethyl) hydrochloride. Elevation of external Ca ions to 20 mM reversibly decreased [Na] by 8±6 mM ( n = 5). Moreover, the chelation of the intracellular Ca with 1,2-bis (2-aminophenoxy) ethane- N,N,N′,N′-tetraacetic acid (BAPTA) exerted an inhibitory action on the NMDG-induced reduction in [Na]. Exposure to 5 mM NaCN for 2 min significantly and reversibly increased [Ca] by 290±37% ( n = 5), but did not affect the [Ca] elevation induced by the NMDG solution. The rise in [Ca] induced by the NMDG solution was not enhanced by ouabain pretreatment. Addition of ouabain did not alter the [Na]. The present results are best explained by the presence of an Na-Ca exchanger in cell membrane and indicate that the activity of Na/K pump is poor in outer hair cells. [ABSTRACT FROM AUTHOR]

The effect of ouabain on the periodic discharge pattern of feline cold receptors was studied in order to substantiate a possible contribution of Na/K pump activity to signal transduction. Afferent activity was recorded from the cold fibres of an isolated preparation of the tongue. The periodic pattern consisted of beating activity and of grouped discharges and was characterized by two parameters, the oscillation frequency and the number of impulses initiated per cycle. Ouabain (10-10 M) induced in all receptors excitatory responses, consisting of a short vigorous increase of activity followed by inhibition. Thus the receptors never stabilized to or maintained a new static level of activity. The ouabain-induced responses occurred repeatedly in several receptors and were produced by remarkable stereotyped modifications of both the oscillation frequency and the number of impulses per cycle. The oscillation frequency attained peak values which increased monotonically with higher static temperatures and which were considerably greater than peak control values. The data indicate that an electrogenic Na/K pump contributes to the transducer process of cold receptors and that inhibition of this pump evidently gives rise to a depolarizing imbalance of the membrane potential, accelerating the oscillation frequency to a maximum value. Thus the oscillation frequency seems to be controlled by temperature and by membrane potential in cold receptors. [ABSTRACT FROM AUTHOR]

To study the stimulating effect of adrenaline (ADR) on active Na/K transport we used double-barrelled ion-sensitive micro-electrodes to measure the activities of extracellular K ( aK) and intracellular Na ( aNa) in isolated preparations of rat soleus muscle, normal human intercostal muscle and one case of hyperkalemic periodic paralysis (h.p.p.). In these preparations bath-application of ADR (10 M) resulted in a membrane hyperpolarization and transient decreases aK and aNa which could be blocked by ouabain (3×10 M). In the h.p.p. muslce a continuous rise of aNa induced by elevation of aK to 5.2 mM could be stopped by ADR. In addition, the intracellular K activity ( aK), the free intracellular Ca concentration ( pCa) and intracellular pH (pH) were monitored in rat soleus muscle. During ADR aK increased, pH remained constant and intracellular Ca apparently decreased. In conclusion, our data show that ADR primarily stimulates the Na/K pump in mammalian skeletal muscle. This stimulating action is not impaired in the h.p.p. muscle. [ABSTRACT FROM AUTHOR]

Using conventional and ion selective microelectrodes, the effect of ouabain (10 mol/l) on peritubular cell membrane potential (PD), on intracellular pH (pH) as well as on the intracellular ion activities of Cl (Cl), K (K), Na (Na) and Ca (Ca) was studied in proximal tubules of the isolated perfused frog kidney. In the absence of ouabain (PD=−57.0±1.9 mV), the electrochemical potential difference of chloride (apparent {ie6-1} and of potassium {ie6-2} is directed from cell to bath, of H {ie6-3}, of Na {ie6-4} and of Ca {ie6-5} from bath to cell. Ouabain leads to a gradual decline of PD, which is reduced to half (PD) within 31±4.6 min (in presence of luminal glucose and phenylalanine), and to a decline of the absolute values of apparent {ie6-6}, of {ie6-7}, {ie6-8} and {ie6-9}. In contrast, an increase of {ei6-10} is observed. At PD apparent Cl increases by 6.2±1.0 mmol/l, pH by 0.13±0.03, Ca by 185±21 nmol/l, and Na by 34.2±4.6 mmol/l, whereas K decreases by 37.7±2.2 mmol/l. The results suggest that the application of ouabain is followed by a decrease of peritubular cell membrane permeability to K, by an accumulation of Ca, Na and HCO in the cell and by a dissipation of the electrochemical Cl gradient. [ABSTRACT FROM AUTHOR]

Stimulation of α-adrenoceptors evokes a different pattern of inotropic responses in atrial and ventricular heart muscle preparations from rats. The inotropic effects are accompanied by different changes in membrane potential. In an attempt to clarify the question whether or to which extent these events are causally related, the effects of phenylephrine on force of contraction, transmembrane potential, Ca current (I) and K currents were comparatively studied in either tissue. In atrial preparations, phenylephrine 10 μmol/l caused an increase in force of contraction, a marked prolongation of the action potential duration and a depolarization of the membrane at rest. In the ventricle, however, the addition of phenylephrine 10 μmol/l produced first a decline in force of contraction associated with a hyperpolarization of the membrane and a reduction in the action potential duration. These changes were followed by an increase in force,of contraction and a slight prolongation of the action potential, whereas the resting membrane potential remained increased. The hyperpolarization was eliminated in the presence of ouabain 100 μmol/l. In enzymatically isolated atrial and ventricular myocytes, the whole-cell voltage clamp technique was used to study membrane currents on exposure to phenylephrine. Phenylephrine 30 μmol/l did not affect the magnitude of I in either cell type. Transient and steady state K outward currents, however, were significantly diminished to a similar extent in atrial and in ventricular myocytes. It is concluded that the positive inotropic effect of α-adrenoceptor stimulation in the rat atrium is related to an increase in action potential duration and a decrease in resting membrane potential due to a decrease in K currents. In the ventricle, phenylephrine additionally activates the Na/K pump thereby hyperpolarizing the membrane. The rapid onset of pump stimulation seems to overwhelm, in the beginning, the phenylephrine-induced decrease in K conductance and therefore to evoke a transient negative inotropic effect. It is assumed that phenylephrine can alter the intracellular Ca concentration due to changes in the action potential duration. The way how Ca 2+ enters the cell remains speculative, since direct changes of Ica were not detected. The more complicated changes in membrane potential in the ventricle suggest that also other mechanisms for the positive inotropic response to phenylephrine must be considered. [ABSTRACT FROM AUTHOR]

All mammalian cells maintain a resting potential generated by ions moving down concentration gradients. In excitable cells, the inside potential is negative relative to outside. In order to maintain this electrochemical gradient, the sodium potassium (Na/K) pump actively transports out three sodium ions for every two potassium ions it brings in. This process generates a net outward current and thus hyperpolaizes the resting potential. I employed dihydroouabain (DHO) to inhibit the Na/K pump and thus measure its contribution to the resting potential. It contributed 9.0 mV at 34°C and 3.8 mV at 25°C. The P/P ratios were calculated at both temperatures before and after subtracting the Na/K pump contribution. These ratios also suggested a decreased contribution of the Na/K pump under hypothermia. Taken together, these results suggest that the pump contribution to the resting potential is more significant at physiologic temperatures (34°C) than at room temperature (25°C), and that estimates of selective permeability can only be accurately obtained after assessing and eliminating the Na/K pump contribution to the resting potential. [ABSTRACT FROM AUTHOR]

We have measured the voltage dependence of the Na/K pump in isolated adult rat cardiac myocytes using the whole-cell patch-clamp technique. In the presence of 1-2 mM Ba and 0.1 m m Cd and nominally Ca-free, Na/K pump current ( I) was measured as the change in current due to 1 mM ouabain. Voltage dependence of Iwas measured between −140 and +40 or +60 mV using square voltage-pulse and voltage-ramp protocols, respectively. With 150 mM extracellular Na (Na) and 5.4 mM extracellular K (K), we found that the Na/K pump shows a strong positive voltage dependence between −140 and 0 mV and is voltage independent at positive potentials. Removing Na reduced the voltage dependence at negative potentials with no effect at positive potentials. When K was reduced, a negative slope appeared in the current-voltage ( I-V) curve at positive potentials. We have investigated whether Na (intracellular Na) might also affect the voltage dependence of Iby varying Na in the patch pipette (Na) between 20 and 85 mM. We found, as expected, that Iincreased markedly as Na was raised, saturating at about 70 mM Na under these conditions. In contast, while Isaturated near +20 mV and declined to about 40% of maximum at −120 mV, there was no effect of Na under these conditions. In contrast, while Isaturated near +20 mV and declined to about 40% of maximum at −120 mV, there was no effect of Na on the voltage dependence of I. This suggests that neither Na binding to the Na/K pump nor the conformational changes dependent on Na binding are voltage dependent. These results are consistent with extracellular ion binding within the field of the membrane but do not rule out the possibility that other steps, such as Na translocation, are also voltage dependent. [ABSTRACT FROM AUTHOR]

Cultured ARL cells respond to abnormally low extracellular K concentrations by increasing the abundance of Na,K-ATPase (the Na/K pump). This response is preceded by significant increases in the mRNAs of the α1 and β1 subunits of this enzyme, implying transcriptional or post-transcriptional regulation in the response. The present study concerned the possible participation of serum factors in low K induction of Na,K-ATPase. In normal K (4.5 m m) or low K (0.68 m m) the presence of 10% calf serum had no effect on Na,K-ATPase activity. The serum independence of the response to low K raised the possibility that low K may itself elicit a 'growth' response. Accordingly, the effect of low K on mRNA abundances of four protooncogenes ( c-fos, c-myc, c-jun and c-ski) was evaluated in the early phase of the response by quantitative Northern blot analysis. The mRNA for c-fos was transiently elevated by low K, with a peak at 30 min. In contrast, low K had no measurable effect on the abundances of c-myc, c-jun and c-ski, for up to 2 hr of exposure. The early elevation of c-fos mRNA makes it a candidate mediator in this signal-transduction pathway. Induction of c-fos mRNA by the phorbol ester, PMA, or by dioctanoyl glycerol, however, had no effect on Na,K-ATPase activity. These results indicate that an increase in c-fos mRNA alone is not sufficient to induce Na,K-ATPase. Whether induction of c-fos is necessary for the response to low K remains to be determined in future studies. [ABSTRACT FROM AUTHOR]

The purpose of this study was to characterize the basolateral membrane of the S3 segment of the rabbit proximal tubule using conventional and ion-selective microelectrodes. When compared with results from S1 and S2 segments, S3 cells under control conditions have a more negative basolateral membrane potential ( V=−69 mV), a higher relative potassium conductance ( t=0.6), lower intracellular Na activity ( A=18.4 mm), and higher intracellular K activity ( A=67.8 mm). No evidence for a conductive sodium-dependent or sodium-independent HCO pathway could be demonstrated. The basolateral Na−K pump is inhibited by 10 m ouabain and bath perfusion with a potassium-free (0-K) solution. 0-K perfusion results in A=64.8 mm, A=18.5 mm, and V=−28 mV. Basolateral potassium channels are blocked by barium and by acidification of the bathing medium. The relative K conductance, as evaluated by increasing bath K to 17 mm, is dependent upon the resting V in both S2 and S3 cells. In summary, the basolateral membrane of S3 cells contains a pump-leak system with similar properties to S1 and S2 proximal tubule cells. The absence of conductive bicarbonate pathways results in a hyperpolarized cell and larger Na and K gradients across the cell borders, which will influence the transport properties and intracellular ion activities in this tubule segment. [ABSTRACT FROM AUTHOR]