Elevation Of Intracellular Na Research Articles

Elevation of Intracellular Na+ Contributes to Expression of Early Response Genes Triggered by Endothelial Cell Shrinkage.

Prolonged hyperosmotic shrinkage evokes expression of osmoprotective genes via nuclear factor NFAT5-mediated pathway and activates Na+ influx via hypertonicity-induced cation channels (HICC). In human umbilical vein endothelial cells (HUVEC) elevation of intracellular sodium concentration ([Na+]i) triggers transcription of dozens of early response genes (ERG). This study examined the role of monovalent cations in the expression of Na+i-sensitive ERGs in iso- and hyperosmotically shrunken HUVEC. Cell volume was measured by 3D reconstruction of cell shape and as 14C-urea available space. Intracellular Na+ and K+ content was measured by flame atomic absorption spectrometry. ERG transcription was estimated by RT-PCR. Elevation of medium osmolality by 150 mM mannitol or cell transfer from hypo- to isosmotic medium decreased cell volume by 40-50%. Hyperosmotic medium increased [Na+]i by 2-fold whereas isosmotic shrinkage had no impact on this parameter. Hyperosmotic but not isosmotic shrinkage increased up-to 5-fold the content of EGR1, FOS, ATF3, ZFP36 and JUN mRNAs. Expression of these ERGs triggered by hyperosmotic shrinkage and Na+,K+-ATPase inhibition by 0.1 µM ouabain exhibited positive correlation (R2=0.9383, p=0.0005). Isosmotic substitution of NaCl by N-methyl-D-glucamine abolished an increment of [Na+]i and ERG expression triggered by mannitol addition. Augmented expression of ERGs in hyperosmotically shrunken HUVEC is mediated by elevation of [Na+]i.

Rebuttal from Marc Pourrier, Sarah Williams, Donald McAfee, Luiz Belardinelli and David Fedida

Papp et al. (2013) raise a number of salient points in disputing the importance of INa,L in diastolic dysfunction in HFpEF. First, they question the selective effect of enhanced INa,L on passive relaxation when one would expect increased INa,L to affect early active relaxation kinetics as well. While the passive phase of relaxation is dependent on diastolic Ca2+ accumulation and Ca2+ extrusion primarily via the Na+–Ca2+ exchanger, the active phase of relaxation, on the other hand is more related to the rate of Ca2+ reuptake into the sarcoplasmic reticulum (SR). Although specific enhancement of INa,L in cardiac myocytes using the sea anemone toxin ATXII slows Ca2+ reuptake into the SR (Sossalla et al. 2008), experimental and clinical data obtained with ranolazine indicate it has mixed effects on active relaxation (Moss et al. 2008; Sossalla et al. 2008; Undrovinas et al. 2010; Coppini et al. 2013; Maier et al. 2013). These discrepancies suggest that the contribution of INa,L to active relaxation may differ in various diseases. Papp et al. also argue that the role of CaMKII in increased INa,L-induced Na+-dependent Ca2+ accumulation in human cardiac myocytes has yet to be demonstrated to be directly associated with diastolic dysfunction and HFpEF. While experiments in human failing myocytes are lacking, there is experimental evidence that associates CaMKII with INa,L, Na+-dependent Ca2+ accumulation and diastolic dysfunction. Specifically, (1) activity of CaMKII is increased in human heart failure (HF) and diastolic dysfunction (Sossalla et al. 2010; Coppini et al. 2013) and (2) CaMKII phosphorylates Na+ channels, resulting in enhanced INa,L, and elevation of intracellular Na+ (Wagner et al. 2006). It is therefore reasonable to consider that CaMKII, as well as other modulators play an important role in INa,L-induced Na+-dependent Ca2+ accumulation and diastolic dysfunction in HFpEF (Zaza et al. 2008). Ranolazine is considered the prototypical INa,L inhibitor, and we agree with Papp et al. that it has other actions able to account for some of the effects observed on diastolic function. However, it is unlikely that the effects of ranolazine on myocardial relaxation are related to pFOX inhibition, as high concentrations (12% inhibition at 100 μm) are required, whereas cardiac function is improved in the presence of ∼10 μm ranolazine. More compelling is the fact that the specific Na+ channel inhibitor tetrodotoxin attenuates INa,L-induced Na+-dependent Ca2+ accumulation in failing ventricular myocytes (Undrovinas et al. 2010). Similarly, the novel highly specific and selective INa,L inhibitor GS967 decreases ATXII-induced increase in intracellular Na+ and diastolic Ca2+ in isolated rabbit ventricular myocytes (Belardinelli et al. 2013).

The Na+/Ca2+ Exchanger Mediates the Effects of Oxidative Stress in Hypertension

Oxidative stress is characterized by the elevation of reactive oxygen species (ROS) including the superoxide anion, hydrogen peroxide and the hydroxyl radical. In almost all animal models of hypertension and humans, reducing oxidative stress with antioxidants will lower blood pressure and elevating oxidative stress increases the blood pressure. Generation of ROS in the renal vasculature is accomplished through NADPH oxidase which is expressed in macula densa and afferent arterioles (AA) and leads to enhanced arteriolar tone and reactivity which precede the onset of hypertension. The underlying mechanism is proposed to involve enhanced tubuloglomeruar feedback (TGF) through depletion of NO through interaction with superoxide anion in the juxtaglomerular apparatus (JGA) to form peroxynitrite and a number of microvascular mechanisms including abnormal elevation of intracellular calcium concentration ([Ca2+]i) which can lead to an increased vascular tone and remodeling. The Na+/Ca2+ exchanger (NCX) is critical in regulating cytosolic calcium homeostasis and our studies show that oxidative stress-induced dysregulation of cytosolic calcium homeostasis is mediated through increased Ca2+ influx through the NCX, an effect which is more pronounced in cells expressing an NCX isoform that is expressed in Salt Sensitive Dahl rat. Ca2+ influx through the NCX is preceded by elevation of intracellular Na+ (Nai) and membrane depolarization. Because of the proximity of the Na+/K+-ATPase and the NCX in the PLasmERosome, it is likely that oxidative stress-induced [Ca2+]i elevation in AA smooth muscle cells is initiated by oxidative stress-induced inhibition of the Na+/K+-ATPase which increases Nai that leads to membrane depolarization and Ca2+ influx through the NCX. Keywords: Oxidative stress, sodium calcium exchanger, hypertension, sodium pump, plasmerosome, reactive oxygen species, superoxide, anion, hydrogen peroxide, NADPH oxidase, blood pressure, tubuloglomeruar feedback, juxtaglomerular apparatus, Na+/Ca2+ exchanger, cytosolic calcium homeostasis, peroxynitrite, cardiovascular disease, nicotine adenine dinucleotide phosphate, super oxide dismutase, neuronal nitric oxide synthase, nitrosylation, tyrosine residues, c-type natriuretic peptide, adrenomedullin, Na-K-ATPase, mesangial cells, NCX1 isoform, vascular smooth muscle cells, sarcoplasmic reticulum, cardiac myocytes, ischemia, hypoxia, myocardial damage, antioxidants, epinephrine, norepinephrine levels, E-ouabain

High glucose inhibits HCO 3 − and fluid secretion in rat pancreatic ducts

Cellular mechanisms underlying the impairment of pancreatic fluid and electrolyte secretion in diabetes were examined using interlobular ducts isolated from rat pancreas. Fluid secretion was assessed by monitoring changes in luminal volume. HCO3(-) uptake across the basolateral membrane was estimated from the recovery of intracellular pH following an acid load. Exposure to high glucose concentrations inhibited fluid secretion and reduced the rate of basolateral HCO3(-) uptake in secretin-stimulated ducts isolated from normal rats. In ducts isolated from streptozotocin-treated diabetic rats, fluid secretion and basolateral HCO3(-) uptake were also severely impaired but could be largely reversed by incubation in normal-glucose solutions. Sodium-dependent glucose cotransporter 1 (SGLT1), glucose transporter (GLUT)1, GLUT2, and GLUT8 transcripts were detected by reverse transcriptase polymerase chain reaction in isolated ducts. Raising the luminal glucose concentration in microperfused ducts caused a depolarization of the membrane potential, consistent with the presence of SGLT1 at the apical membrane. Unstimulated ducts filled with high-glucose solutions lost luminal fluid by a phlorizin-sensitive mechanism, indicating that pancreatic ducts are capable of active glucose reabsorption from the lumen via SGLT1. In ducts exposed to high glucose concentrations, continuous glucose diffusion to the lumen and active reabsorption via SGLT1 would lead to elevation of intracellular Na+ concentration and sustained depolarization of the apical membrane. These two factors would tend to inhibit the basolateral uptake and apical efflux of Cl(-) and HCO3(-) and could therefore account for the impaired fluid and electrolyte secretion that is observed in diabetes.

What regulates Na+/Ca2+ exchange? Focus on “Sodium-dependent inactivation of sodium/calcium exchange in transfected Chinese hamster ovary cells”

### The Necessity for NCX1 Regulation Considering that the cardiac Na+/Ca2+ exchanger (NCX1.1) is the predominant mechanism for transsarcolemmal Ca2+ efflux in cardiac myocytes, it is obvious that this transport system must be regulated actively. Sodium/calcium exchange removes roughly the same

Role of Hyperforin in the Pharmacological Activities of St. John's Wort

The phloroglucinol derivative hyperforin has been recently shown to be a major antidepressant component in the extract of Hypericum perforatum. Experimental studies clearly demonstrated its activity in different behavioral models of depression. Moreover clinical studies linked the therapeutic efficacy of Hypericum extracts to their hyperforin content, in a dose-dependent manner. The molecular mechanism of action of hyperforin is still under investigation. Hyperforin has been shown to inhibit, like conventional antidepressants, the neuronal uptake of serotonin, norepinephrine and dopamine. However, hyperforin inhibits also the uptake of gamma-aminobutyric acid (GABA) and L-glutamate. The uptake inhibition by hyperforin does not involve specific binding sites at the transporter molecules; its mechanism of action seems to be related to sodium conductive pathways, leading to an elevation in intracellular Na(+) concentration. Other additional mechanisms of action of hyperforin, involving ionic conductances as well synaptosomal and vesicular function, have been suggested. In addition to its antidepressant activity, hyperforin has many other pharmacological effects in vivo (anxiolytic-like, cognition-enhancing effects) and in vitro (antioxidant, anticyclooxygenase-1, and anticarcinogenic effects). These effects could be of clinical importance. On the other hand, the role of hyperforin in the pharmacological interactions occurring during Hypericum extract therapy must be fully investigated. Hyperforin seems to be responsible for the induction of liver cytochrome oxidase enzymes and intestinal P-glycoprotein. Several pharmacokinetic studies performed in rats and humans demonstrated oral bioavailability of hyperforin from Hypericum extract. Only recently a new chromatographic method for detection of hyperforin in the brain tissue has been developed and validated. Taking into account the chemical instability of hyperforin, current efforts are directed to the synthesis of new neuroactive derivatives.

Plasma membrane electron transport coupled to Na + extrusion in the halotolerant alga Dunaliella

The halotolerant alga Dunaliella adapts to exceptionally high salinity and maintains low [Na +] in at hypersaline solutions, suggesting that it possesses efficient mechanisms for regulating intracellular Na +. In this work we examined the possibility that Na + export in Dunaliella is linked to a plasma membrane electron transport (redox) system. Na + extrusion was induced in Dunaliella cells by elevation of intracellular Na + with Na +-specific ionophores. Elevation of intracellular Na + was found to enhance the reduction of an extracellular electron acceptor ferricyanide (FeCN). The quinone analogs NQNO and dicumarol inhibited FeCN reduction and led to accumulation of Na + by inhibition of Na + extrusion. These inhibitors also diminished the plasma membrane potential in Dunaliella. Anaerobic conditions elevated, whereas FeCN partially decreased intracellular Na + content. Cellular NAD(P)H level decreased upon enhancement of plasma membrane electron transport. These results are consistent with the operation of an electrogenic NAD(P)H-driven redox system coupled to Na + extrusion in Dunaliella plasma membrane. We propose that redox-driven Na + extrusion and recycling in Dunaliella evolved as means of adaptation to hypersaline environments.

Inhibition of different pathways influencing Na + homeostasis protects organotypic hippocampal slice cultures from hypoxic/hypoglycemic injury

A prominent feature of cerebral ischemia is the excessive intracellular accumulation of both Na + and Ca 2+, which results in subsequent cell death. A large number of studies have focused on pathways involved in the increase of the intracellular Ca 2+ concentration [Ca 2+] i, whereas the elevation of intracellular Na + has received less attention. In the present study we investigated the effects of inhibitors of different Na + channels and of the Na +/Ca 2+ exchanger, which couples the Na + to the Ca 2+ gradient, on ischemic damage in organotypic hippocampal slice cultures. The synaptically evoked population spike in the CA1 region was taken as a functional measure of neuronal integrity. Neuronal cell death was assessed by propidium iodide staining. The Na + channel blocker tetrodotoxin, and the NMDA receptor blocker MK 801, but not the AMPA/kainate receptor blocker NBQX prevented ischemic cell death. The novel Na +/Ca 2+ exchange inhibitor 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), which preferentially acts on the reverse mode of the exchanger, leading to Ca 2+ accumulation, also reduced neuronal damage. At higher concentrations, KB-R7943 also inhibits Ca 2+ extrusion by the forward mode of the exchanger and exaggerates neuronal cell death. Neuroprotection by KB-R7943 may be due to reducing the [Ca 2+] i increase caused by the exchanger.

Stimulus interval-dependent differences in Ca2+ transients and contractile responses of diabetic rat cardiomyocytes.

The aim of this study was to gain further insights into the consequences of insulin-dependent diabetes mellitus on cardiomyocyte calcium handling. The effects of steady state and transient changes in stimulus frequency on the intracellular Ca2+ transient and cell shortening were examined in left ventricular cardiomyocytes isolated from the hearts of control and streptozotocin-induced diabetic rats. During steady state stimulation diabetic rat cardiomyocytes displayed a slower decay of the Ca2+ transient and longer times for maximum cell shortening and re-lengthening. At 1.5 mM extracellular [Ca2+], increasing stimulus frequency over the range 0.2-1.0 Hz led to an increase in resting and peak [Ca2+]i as well as the amplitude of the transient in both the control and diabetic groups. At frequencies greater than 0.4 Hz the amplitude of the transient was significantly depressed in diabetic rat cells and this was not normalized by increasing extracellular [Ca2+] to 2.5 mM. Recovery of sarcoplasmic reticulum (SR) Ca2+ release was measured from the time course of restitution of the intracellular Ca2+ transient. In both control and diabetic rat cardiomyocytes recovery of the transient occurred in two phases. In diabetic rat myocytes, the initial rapid phase of restitution at intervals <1 s was markedly slowed. The fraction of Ca2+ recirculating between the SR and the cytosol was estimated from the decline in amplitude of transients following post-rest potentiation. There was no difference in this fraction between control and diabetic rat cells either at 1.5 or 2.5 mM extracellular [Ca2+]. The blunted frequency response of diabetic rat cardiomyocytes at frequencies greater than 0.4 Hz is consistent with reduced SR Ca2+ uptake leading to reduced SR Ca2+ content and subsequent release. At stimulus intervals greater than 1 Hz this is likely to be exacerbated by slower recovery of SR Ca2+ release. Despite the evidence for depressed SR Ca2+ uptake, the relative amount of Ca2+ recirculating within diabetic rat cardiomyocytes remains unaltered. This is most likely due to an accompanying reduction in Ca2+ efflux from the cell due either to depressed Na+/Ca2+ exchanger activity, or an elevation in intracellular Na+ levels.

Inhibitors of Na+/Ca2+ exchanger prevent oxidant-induced intracellular Ca2+ increase and apoptosis in a human hepatoma cell line

Oxidative stress appears to be implicated in the pathogenesis of various diseases including hepatotoxicity. Although intracellular Ca2+ signals have been suggested to play a role in the oxidative damage of hepatocytes, the sources and effects of oxidant-induced intracellular Ca2+ increases are currently debatable. Thus, in this study we investigated the exact source and mechanism of oxidant-induced liver cell damage using HepG2 human hepatoma cells as a model liver cellular system. Treatment with 200 μM of tert-butyl hydroperoxide (tBOOH) induced a sustained increase in the level of intracellular reactive oxygen intermediates (ROI) and apoptosis, assessed by 2′,7′-dichlorofluorescein fluorescence and flow cytometry, respectively. Antioxidants, N-acetyl cysteine (NAC) or N,N′-diphenyl-p-phenylenediamine significantly inhibited both the ROI generation and apoptosis. In addition, tBOOH induced a slow and sustained increase in intracellular Ca2+ concentration, which was completely prevented by the antioxidants. An intracellular Ca2+ chelator, bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid/cetoxymethyl ester significantly suppressed the tBOOH-induced apoptosis. These results imply that activation of an intracellular Ca2+ signal triggered by increased ROI may mediate the tBOOH-induced apoptosis. Both intracellular Ca2+ increase and induction of apoptosis were significantly inhibited by an extracellular Ca2+ chelator or Na+/Ca2+ exchanger blockers (bepridil and benzamil), whereas neither Ca2+ channel antagonists (verapamil and nifedipine) nor a nonselective cation channel blocker (flufenamic acid) had an effect. These results suggest that tBOOH may increase intracellular Ca2+ through the activation of reverse mode of Na+/Ca2+ exchanger. However, tBOOH decreased intracellular Na+ concentration, which was completely prevented by NAC. These results indicate that ROI generated by tBOOH may increase intracellular Ca2+ concentration by direct activation of the reverse mode of Na+/Ca>2+ exchanger, rather than indirect elevation of intracellular Na+ levels. Taken together, these results suggest that the oxidant, tBOOH induced apoptosis in human HepG2 cells and that intracellular Ca2+ may mediate this action of tBOOH. These results further suggest that Na+/Ca2+ exchanger may be a target for the management of oxidative hepatotoxicity.

Metabolism of trout red blood cells: correlation between cation transport and oxygen uptake following adrenergic stimulation

Red blood cells from rainbow trout rely mainly on oxidative phosphorylation to cover the energy demand of processes such as cation transport and protein synthesis. Stimulation of these cells with catecholamines leads to an increase in the intracellular Na + concentration due to adrenergic activation of the erythrocyte Na +–H + exchanger. Adrenergic stimulation also increases red cell oxidative phosphorylation which largely can be ascribed to increased activity of the Na +–K + pump in response to the increase in intracellular Na + concentration. Based on experiments with the Na +-ionophore, monensin, it has been suggested that adrenergic stimulation is a prerequisite for the increase in oxidative ATP-production observed when intracellular Na + is increased. Using washed red blood cells from rainbow trout, the present study examines the role for adrenergic stimulation in the metabolic response when intracellular Na + is elevated. It is shown that while monensin (5×10 −8–5×10 −6 M) causes a persistent up-to-12-fold increase in the intracellular Na + concentration, it only transiently elevates the activity of the Na +–K + pump. Furthermore, monensin (5×10 −6 M) decreased rather than increased red cell oxygen uptake. In contrast, adrenergic stimulation, which produced a 4-fold elevation in intracellular Na +, led to a 2.5-fold increase in red cell oxygen uptake and an 11-fold increase in Na +–K + pump activity. Forty percent of the increase in oxygen uptake was blocked by inhibition of the Na +–K + pump. In monensin-treated cells, adrenergic stimulation was, however, without effect on oxygen uptake and Na +–K + pump activity. These data do not support the theory that adrenergic stimulation has a direct effect on the ATP-producing processes of trout red blood cells but rather suggest that the effect of catecholamines on oxygen uptake is restricted to effects on the energy-consuming processes of the cells.

Elevation of intracellular Na+ induced by hyperpolarization at the dendrites of pyramidal neurones of mouse hippocampus.

1. Whole-cell recordings were made from CA1 pyramidal cells in mouse hippocampal slices with patch pipettes containing the sodium indicator dye SBFI (sodium binding benzofuran isophthalate). Using a high-speed imaging system, we investigated changes in intracellular sodium concentration, [Na+]i, in response to hyperpolarizing pulses applied to the soma. 2. In current-clamp recordings, we detected increases in [Na+]i during negative current injection. Hyperpolarization-induced [Na+]i elevation was more prominent in the middle apical dendrites than in the soma. 3. In the voltage-clamp mode, hyperpolarization induced rapid increases in [Na+]i at the apical dendrites that were significantly faster than those at the soma. The signals were not affected by bath application of 1 microM TTX, but were reduced by 5 mM CsCl. 4. Changes in membrane potential recorded from the apical dendrites in response to negative currents were significantly smaller than those recorded from the soma. In the presence of 5 mM CsCl, the I-V relationships measured at the soma and the dendrites became almost identical, indicating that CsCl-sensitive components are predominantly in the apical dendrites. 5. These results suggest that hyperpolarization-induced [Na+]i elevations reflect Na+ influx through the non-selective cation channel (Ih channel), and that this channel is distributed predominantly in the apical dendrites. The non-uniform Na+ influx may contribute to integrative functions of the dendrites.

Effect of Na + on Na +,K +-ATPase α-subunit expression and Na +-pump activity in aortic smooth muscle cells

In earlier studies we demonstrated that cyclical mechanical strain on vascular smooth muscle cells increases intracellular Na + and upregulates the α-1 and α-2 isoform expression of Na +,K +-ATPase, and that the increase of intracellular Na + and upregulation of the α-2 isoform expression are blocked by Gd 3+, which blocks entry of ions (including Na +) through stretch-activated channels. The present study was designed to investigate the role of intracellular Na + in Na +,K +-ATPase regulation by increasing intracellular Na + with chronic ouabain treatment. In parallel experiments, we measured Na +,K +-ATPase α isoform expression, Na +-pump activity and intracellular Na + in cultured aortic smooth muscle cells after treatment with two concentrations of ouabain for various time periods. Treatment with 100 nM ouabain resulted in a significant elevation in intracellular Na + after 1 (21%) and 2 h (12%), but the value returned to baseline after 12 h. Both α-1 and α-2 subunits of Na +,K +-ATPase were significantly upregulated after 1 through 4 days. Na +-pump activity was also stimulated, and the time course of this effect closely followed protein expression. At 200 μM of ouabain, the effects on intracellular Na +, isoform expression and Na +-pump activity at earlier time points (1 h through 1 day) were similar to those with 100 nM treatment, but prolonged treatment (2 and 4 days) resulted in an accumulation of intracellular Na + and inhibition of the isoform expression and Na +-pump activity, possibly due to general dysfunction of the cells as a result of chronic exposure to high concentrations of ouabain. We conclude that elevated intracellular Na + can serve as a signal to mediate the α isoform upregulation and the regulatory process requires less than one day.

Estimation of Na-blocking efficiency of rihlocaine and its combinations with low-molecular-weight polymers on isolated rat cardiomyocytes

The effect of glucose-base polymers (dextrans with relative molecular weight 10, 40, and 70 kD) and vinylpyrrolidone (polyvinylpyrrolidone: 10, 24, and 40 kD) on changes in the sarcoplasmic Na+ concentration in stimulated cardiomyocytes (1.0 Hz, 10 msec, 60 mV) were examined. In the concentration range of 1–100 μM, the polymer preparations produced cardioprotective effect on cells incubated under hypoxic conditions; the effect depended on the nature and molecular weight of the polymer. Rihlocaine (25 μM) inhibits by 42% elevation of intracellular Na+ induced by plasma membrane depolarization. Dextran 40 is shown to significantly increase Na-blocking effect of rihlocaine.

A possible role of the ATP-sensitive potassium ion channel in determining the duration of spike-bursts in mouse pancreatic β-cells

The pancreatic β-cell displays an electrical activity consisting of spike bursts and silent phases at glucose concentrations of about 10 mM. The mechanism of initial depolarization induced by glucose is well defined. However, the mechanism inducing the silent phase has not been fully elucidated. In the present study, the possibility of involvement of ATP-sensitive K + channels in repolarization was examined using the patch-clamp technique in the cell-attached recording configuration. Ouabain (0.1 mM), an inhibitor of Na +/K +-ATPase, caused a complete suppression of ATP-sensitive K + channel activity followed by typical biphasic current deflections, which were due to action potentials. The channel activity was also inhibited by removal of K + from a perifusion solution. Furthermore, the activity of ATP-sensitive K + channels was markedly inhibited either by replacement of external NaCl with LiCl or by addition of amiloride (0.2 mM), a blocker of Na +/H + antiport. Addition of L-type Ca 2+ channel blockers such as Nifedipine or Mn 2+ induced the complete suppression of K + channel activity. These findings strongly suggest that a fall in ATP consumption results in sustained depolarization, and that the repolarizations interposed between spike-bursts under normal ionic conditions are due to the periodical fall of ATP concentration brought about by periodical acceleration of ATP consumption at Na +/K +-pumps. It is concluded that the elevation of intracellular Na + concentration as a consequence of accelerated Na +/Ca 2+-countertransport during the period of spike-burst enhances ATP consumption, leading to a fall in ATP concentration which is responsible for termination of spike-burst and initiation of repolarization.

Action of insulin on Na(+)-K(+)-ATPase and the Na(+)-K(+)-2Cl- cotransporter in 3T3-L1 adipocytes

The Na(+)-K(+)-ATPase presents several different isoforms of its alpha- and beta-subunits. We detected alpha 1- and beta 1-mRNA transcripts and polypeptides in 3T3-L1 fibroblasts; during differentiation into adipocytes, alpha 1-mRNA decreased, alpha 2-mRNA was induced, beta 1-mRNA dropped to undetectable levels, and beta 2-mRNA was never expressed, suggesting that 3T3-L1 adipocytes may express an unidentified Na(+)-K(+)-ATPase beta-subunit isoform. Insulin rapidly increased ion pump activity [ouabain-sensitive 86Rb+(K+) uptake] in 3T3-L1 fibroblasts and adipocytes without changing the plasma membrane concentration of alpha 1- or alpha 2-subunits as determined by subcellular membrane fractionation and immunoblotting or by [3H]ouabain binding to intact cells. Monensin, which raises the concentration of intracellular Na+, increased Na(+)-K+ pump activity, and no further stimulation was achieved with insulin. The stimulation of the pump by insulin was reduced by bumetanide, an inhibitor of the Na(+)-K(+)-2Cl- cotransporter, and was prevented by omission of extracellular Cl-. Insulin increased both ouabain-sensitive and bumetanide-sensitive 86Rb+(K+) uptake. These results suggest that insulin activation of the Na(+)-K(+)-ATPase in 3T3-L1 adipocytes is mediated by an elevation in intracellular Na+ that is likely the consequence of Na(+)-K(+)-2Cl- cotransporter activation.

Effect of ouabain on relaxation induced by cromakalim in human and canine mesenteric arteries

We investigated the effect of cromakalim, a K + channel opener, that activates indirectly the Na +-K + pump, in association with increased K + conductance in the mesenteric arteries. In 65% of human mesenteric arteries tested, the concentration-dependent relaxation curves for cromakalim were biphasic: the low concentration (<10 −7 M) effect was preferentially inhibited by ouabain, whereas the higher concentration effect was significantly inhibited by glibenclamide. In branches of canine mesenteric artery, the cromakalin-induced relaxation was inhibited by pretreatment with ouabain (1 μM) as well as by glibenclamide (1 μM). The reduction in contraction of human and canine mesenteric arterial strips caused by cromakalim was totally reversed by pretreatment with ouabain (1 μM) or glibenclamide (1 μM). On the other hand, in canine mesenteric artery, cromakalim caused a significant stimulation of 22Na + influx and ouabain-sensitive 86Rb + uptake in association with increased 86Rb + efflux, all of which were inhibited by glibenclamide (1 μM). Thus, it is suggested that cromakalim possesses the additional property to stimulate the Na +-K + pump through an elevation in intracellular Na +, resulting in strong relaxation of blood vessels.

Na(+)-Ca2+ exchange in cultured rat hepatocytes: evidence against a role in cytosolic Ca2+ regulation or signaling

Plasma membrane Na(+)-Ca2+ exchange contributes importantly to the regulation of cytosolic Ca2+ concentration ([Ca2+]i) in excitable cells. Despite extensive study in excitable tissues, the role of this transporter in the regulation of [Ca2+]i in hepatocytes is unknown, and conflicting information has been reported regarding the presence of Na(+)-Ca2+ exchange in hepatocyte plasma membrane vesicles. We have therefore assessed the role of Na(+)-dependent Ca2+ transport in the regulation of [Ca2+]i in rat hepatocytes in primary culture under basal conditions and after exposure to vasopressin, a hormone that elevates [Ca2+]i. Ca2+ efflux, measured using 45Ca, did not differ in the presence or absence of extracellular Na+, either under basal conditions or in response to vasopressin. [Ca2+]i, measured using the Ca2(+)-sensitive dye fura-2, was not altered by transient or prolonged exposure to Na(+)-free media or by exposure to ouabain in concentrations sufficient to produce a five-fold elevation in intracellular Na+ concentration. The [Ca2+]i response to vasopressin was also unaffected by Na+ removal or ouabain. By contrast, in cultured rat cardiac myocytes, cells that possess Na(+)-Ca2+ exchange, transient or prolonged Na+ removal as well as ouabain exposure produced greater than fivefold increases in [Ca2+]i compared with controls. We conclude that Na(+)-Ca2+ exchange does not contribute to the regulation of [Ca2+]i in hepatocytes.

Nucleated red cell function: metabolism and pH regulation

The full extent and apportionment of aerobic and anaerobic contributions to energy transduction for membrane pumps associated with cellular pH regulation are very poorly understood. One way of approaching this problem at the cellular level is by using the nucleated erythrocyte as a model cell. Indeed, the aerobic and anaerobic capacity of salmonid erythrocytes and their β-adrenergic mediated pH regulation offers a model "pH regulating system" for examining cellular strategies of response to acute and (or) chronic changes in oxygen availability. Much of our work has focused on the balance between metabolic energy production and the maintenance of erythrocytic pH through primarily or secondarily active ionic exchange mechanisms at the cell membrane. Upon adrenergic stimulation, a rise in cyclic AMP activates the Na+–H+ exchanger, leading to cell alkalinization and an elevation of intracellular Na+. The increased Na+ evidently stimulates Na+,K+-ATPase activity and the increased ATP consumption is matched with aerobic energy production. The pHi that is subsequently established appears to be set by levels of poly anionic phosphates.

Activation and proliferation signals in murine macrophages: Stimulation of Na+, K+‐ATPase activity by hemopoietic growth factors and other agents

Purified colony stimulating factor (CSF-1) stimulates the Na+,K+-ATPase activity of murine bone marrow-derived macrophages (BMM) and resident peritoneal macrophages (RPM) measured as ouabain-sensitive 86Rb+ uptake. Similar concentrations of CSF-1 stimulate the Na+,K+-ATPase activity and DNA synthesis in BMM whilst ouabain, a specific inhibitor of the Na+,K+-ATPase, also inhibits this CSF-1-mediated DNA synthesis. Other purified hemopoietic growth factors, granulocyte-macrophage CSF (GM-CSF) and interleukin-3 (IL-3), and the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), even though differing in their mitogenic capabilities, are also stimulators of the Na+,K+-ATPase activity in BMM and RPM. The non-mitogenic agents, lipopolysaccharide (LPS) and Concanavalin A (Con A), are also active. CSF-1 stimulation of the Na+,K+-ATPase was shown to be dependent on elevation of intracellular Na+ via an amiloride sensitive Na+-channel, most likely representing Na+/H+ exchange activity. Such stimulation of Na+,K+-ATPase activity via activation of the Na+/H+ exchange appears to be a necessary but insufficient early macrophage response for subsequent DNA synthesis.