Pathways of Rb+ influx and their relation to intracellular [Na+] in the perfused rat heart. A 87Rb and 23Na NMR study

The aims of this study were to characterize the routes of influx of the K+ congener, Rb+, into cardiac cells in the perfused rat heart and to evaluate their links to the intracellular Na+ concentration ([Na+]i) using 87Rb and 23Na nuclear magnetic resonance (NMR) spectroscopy. The rate constant for Rb+ equilibration in the extracellular space was 8.5 times higher than that for the intracellular space. The sensitivity of the rate of Rb+ accumulation in the intracellular space of the perfused rat heart to the inhibitors of the K+ and Na+ transport systems has been analyzed. The Rb+ influx rates were measured in both beating and arrested hearts: both procaine (5 mmol/L) and lidocaine (1 mmol/L) halved the Rb+ influx rate. In procaine-arrested hearts, the Na+,K(+)-ATPase inhibitor ouabain (0.6 mmol/L) decreased Rb+ influx by 76 +/- 24% relative to that observed in untreated but arrested hearts. Rb+ uptake was insensitive to the K+ channel blocker 4-aminopyridine (1 mmol/L). The inhibitor of Na+/K+/2 Cl- cotransport bumetanide (30 mumol/L) decreased Rb+ uptake only slightly (by 9 +/- 8%). Rb+ uptake was dependent on [Na+]i: it increased by 58 +/- 34% when [Na+]i was increased with the Na+ ionophore monensin (1 mumol/L) and decreased by 48 +/- 9% when [Na+]i was decreased by the Na+ channel blockers procaine and lidocaine. Dimethylamiloride (15 to 20 mumol/L), an inhibitor of the Na+/H+ exchanger, slightly reduced [Na+]i and Rb+ entry into the cardiomyocytes (by 15 +/- 5%). 31P NMR spectroscopy was used to monitor the energetic state and intracellular pH (pHi) in a parallel series of hearts. Treatment of the hearts with lidocaine, 4-aminopyridine, dimethylamiloride, or bumetanide for 15 to 20 minutes at the same concentrations as used for the Rb+ and Na+ experiments did not markedly affect the levels of the phosphate metabolites or pHi. These data show that under normal physiological conditions, Rb+ influx occurs mainly through Na+,K(+)-ATPase; the contribution of the Na+/K+/2 Cl- cotransporter and K+ channels to Rb+ influx is small. The correlation between Rb+ influx and [Na+bdi during infusion of drugs that affect [Na+]i indicates that, in rat hearts at 37 degrees C, Rb+ influx can serve as a measure of Na+ influx. We estimate that, at normothermia, at least 50% of the Na+ entry into beating cardiac cells is provided by the Na+ channels, with only minor contributions (< 15%) from the Na+/K+/2 Cl- cotransporter and the Na+/H+ exchanger.     

ATP1AL1, a member of the non-gastric H,K-ATPase family, functions as a sodium pump

The human ATP1AL1-encoded protein (an alpha subunit of the human non-gastric H,K-ATPase) has previously been shown to assemble with the gastric H,K-ATPase beta subunit (gH,Kbeta) to form a functionally active ionic pump in HEK 293 cells. This pump has been found to be sensitive to both SCH 28080 and ouabain. However, the 86Rb+-influx mediated by the ATP1AL1-gH,Kbeta heterodimer in HEK 293 cells is at least 1 order of magnitude larger than the maximum ouabain-sensitive proton efflux detected in the same cells. In this study we find that the intracellular Na+ content in cells expressing ATP1AL1 and gH,Kbeta is two times lower than that in control HEK 293 cells in response to incubation for 3 h in the presence of 1 microM ouabain. Moreover, analysis of net Na+ efflux in HEK 293 expressing the ATP1AL1-gH,Kbeta heterodimer reveals the presence of Na+ extrusion activity that is not sensitive to 1 microM ouabain but can be inhibited by 1 mM of this drug. In contrast, ouabain-inhibitable Na+ efflux in control HEK 293 cells is similarly sensitive to either 1 microM or 1 mM ouabain. Finally, 86Rb+ influx through the ATP1AL1-gH,Kbeta complex is comparable to the 1 mM ouabain-sensitive Na+ efflux in the same cells. The data presented here suggest that the enzyme formed by ATP1AL1 and the gastric H,K-ATPase beta subunit in HEK 293 cells mediates primarily Na+,K+ rather than H+,K+ exchange.     

Ouabain-sensitive ion fluxes in the smooth muscle of the guinea-pig's taenia coli

1. Tissues with raised intracellular Na levels, produced by incubation in K-free media, were used throughout. The uptake of 42K by these Na-loaded tissues was followed for 10 min in the presence and absence of 1-37 X 10(-4) M ouabain, this being sufficient to inhibit Na pumping maximally. Subtraction of the uptake seen in the presence from that seen in the absence of ouabain gave estimates of the pumped ouabain-sensitive K uptake. 2. In Na-free (MgCl2) medium this depended on the [K]0 in a sigmoidal fashion with a half maximal [K]0 for activation of some 4mM. The maximal uptake of K was 3 m-mole/kg.min corresponding to a transmembrane flux of some 12-5 p-mole. cm-2.sec-1. 3. In the presence of Na the K activation curve became more obviously sigmoid and higher concentrations of K were needed to achieve a given active K influx. The results were well fitted by assuming that Na and K competed for two identical, non-interacting sites on the external pump face. 4. Addition of K during the efflux of 24Na into a Na-free (MgCl2) medium led to an increased rate of tracer loss. The magnitude of this increase depended on the [K] used in a hyperbolic fashion and it was abolished by addition of ouabain. The [K] causing half-maximal activation of ouabain-sensitive Na efflux was in the order of 1-2 mM. 5. When the [K] in the uptake media was 1-5 mM; Na, Li, Rb and Cs all inhibited ouabain-sensitive K uptake, the order of effectiveness being Rb greater than Cs greater than Na greater than Li. With a E1TKA10 OF 0-15 MM low concentrations of Cs and Rb were shown to stimulate K uptake. Such an effect is predicted by assuming two ion binding sites on the pump's outer face, and that the pump can translocate mixtures of K and either Rb or Cs...     

Reduced myocardial Na+, K(+)-pump capacity in congestive heart failure following myocardial infarction in rats

We examined changes in expression and function of the cardiac Na+, K(+)-pump in a post-infarction rat model of hypertrophy and congestive heart failure (CHF). Myocardial infarction was induced by ligation of the left coronary artery in Wistar rats and hearts were obtained from animals with CHF and from sham operated rats after 6 weeks. In the CHF group the ratio of heart weight to body weight was 70% greater compared to sham (*P < 0.05) and all left-ventricular end-diastolic pressures (LVEDP) were above 15 mmHg. The expression of the alpha 1- and beta 1-subunits (mRNA and protein) of the Na+, K(+)-pump was not significantly different in CHF and sham. As compared to sham the alpha 2 isoform, mRNA and protein levels were lower in CHF hearts by 25 and 55%, respectively, whereas the alpha 3 isoform mRNA was greater by 120% in CHF. The alpha 3 protein was not detectable in sham but a prominent band was seen in CHF. Cell volume of isolated cardiomyocytes was 30% larger in CHF. Cardiomyocytes containing the Na+ sensitive fluorescent dye SBFI were loaded to an intracellular Na+ concentration ([Na+]i] of about 140 mM in a K(+)- and Mg(2+)-free medium (140 mM Na+, free Ca2+ of 10(-8) M). To avoid back leak of Na+ and to ensure no voltage effects on the Na+, K(+)-pump extracellular Na+ was subsequently removed, and 6 mM Mg2+ was added to the superfusate, The Na+, K(+)-pump was then reactivated by 10 mM Rb+. SBFI fluorescence ratio decreased mono-exponentially with a time constant (tau) of 191 +/- 15 s in sham (n = 8) and 320 +/- 38 s in CHF (n = 9) rats (P < 0.01). These changes in fluorescence indicate that the maximum rate of decline of [Na+]i from 100 to 35 mM was 39% (P < 0.005) slower in CHF compared to sham, whereas maximum pump rate per cell was not significantly altered (9.0 +/- 0.7 fmol/s in sham and 7.1 +/- 0.7 fmol/s in CHF cells). The [Na+]i which caused 50% pump activation (k0.5) was also not altered in CHF (40 mM in both groups). We conclude that the number of Na+, K(+)-pumps per cell was maintained in CHF but an isoform switch of the alpha 3-replacing the alpha 2-isoform occurred. However, maximum Na+, K(+)-pump rate in terms of rate of change of [Na+]i was significantly attenuated in CHF, most likely as a result of increased cell size.     

Decrease in the ratio of high- to low-affinity isozymes of (Na+ +K+)-ATPase during the development of rat cardiac ventricles

The ratio of (Na+ +K+)-ATPase (EC 3.6.1.3.)isoforms with high and low affinity for cardiac glycosides was studied in heart preparations from neonatal, 3-month and 6-month old Wistar rats. Biphasic ouabain inhibition curves of (Na+ +K+)-ATPase activity indicated that the relative contribution of the high-affinity process decreased from 34% at 9 days to 23% at 3 months and to 10% at 6 months. Scatchard plots for [3H]ouabain binding were curvilinear and indicated that the relative contribution of the high-affinity sites (Kd = 0.1-0.25 microM) decreased by about one-half between 3 months (19-24%, N = 2) and 6 months (9-11%, N = 2).     

Ryanodine binding sites measured in small skeletal muscle biopsies

A method allowing measurement of the concentration of [3H]ryanodine binding sites in small skeletal muscle specimens (> 10-20 mg) was developed. A membrane fraction containing 87% of the [3H]ryanodine binding sites of the tissue and exhibiting one single KD of 18-27 nmol l-1 in rat and 8 nmol l-1 in human muscles (p < 0.05) was obtained. Maximum binding to rat EDL and soleus muscles equalled 59.1 and 16.2 pmol g-1 wet wt, whereas in human gluteus muscles binding was 12.3 pmol g-1 wet wt. The [3H]ryanodine binding showed a dependency on Mg2+ and pH similar to previously published results. As measured by Ca2+ selective mini-electrodes, the [Ca2+] causing 50% of maximum [3H]ryanodine binding (K0.5) was 200-400 nmol l-1 for different muscles. [Ca2+] higher than 1 mmol l-1 caused strong inhibition of the [3H]ryanodine binding, and both high and low [Ca2+] caused rapid dissociation of the complex. At ionic strength lower than 100 mmol l-1, more than 50% of the [3H]ryanodine was bound to particles with size less than 1.2 microns which were not retained by GF/C filters. Thus, we have obtained an almost complete quantitative recovery of functional RyRs from small muscle specimens exhibiting high affinity for Ca2+, which stimulated ligand binding.     

Serotonin modulation of low-affinity ouabain binding in rat brain determined by quantitative autoradiography

Previous results showed that Na+/K+-ATPase may have a functional relationship with the neurotransmitter serotonin which activates the glial sodium pump in the rat brain. Both the reaction rate (V) of Na+/K+-ATPase activity and [3H]ouabain binding were significantly increased in the presence of serotonin. It is not known, however, which alpha isoform is involved in the Na+/K+-ATPase response to serotonin and its regional distribution. Quantitative autoradiography of [3H]ouabain binding to rat brain slices was employed at different [3H]ouabain concentrations in order to gain information on both the distribution and the possible isoform involved. The results showed that 1500 nM [3H]ouabain binding was sensitive to serotonin 10(-3) M and significantly increased in the following brain regions: frontal cortex, areas CA1, CA2, and CA3 of the hippocampus, presubiculum, zona incerta, caudate putamen and the amygdaloid area, confirming and extending previous results. An effect of serotonin on brain but not kidney tissue at high, 1500 nM, and the lack of effect at low, 50 nM [3H]ouabain concentrations, strongly suggests the participation of the alpha2 isoform in the response of the pump to the neurotransmitter. Glial cells showed stimulation of ouabain binding by serotonin at ouabain concentrations above 350 nM. The present results open interesting questions related to the brain regions involved and the K+ handling by the glial alpha2 isoform of the pump.     

Opioid tolerance in neonates: mechanisms, diagnosis, assessment, and management

Na+,K+-ATPase activity of rat brain synaptosomal membranes was evaluated in the presence of an inhibitory fraction II-E (termed endobain E), isolated by gel filtration and anionic exchange HPLC of a rat brain soluble fraction. We studied endobain E aging, analyzed its inhibitory potency in the absence or presence of ouabain as well as its ability to block high affinity [3H]ouabain binding to cerebral cortex membranes. Similar loss of endobain E activity was observed when samples were stored either dried or in solution. Endobain E fraction inhibited synaptosomal membrane Na+,K+-ATPase activity in a concentration-dependent manner and the slope of the corresponding curve strongly resembled that of ouabain. Assays performed in the presence of endobain E and ouabain indicated that the inhibitory effect was additive or less than additive, depending on their respective concentrations during preincubation and/or incubation. High affinity [3H]ouabain binding to cerebral cortex membranes proved concentration-dependent from 0.10 to 0.50 mg protein per ml; binding inhibition by endobain E was independent of protein concentration within the above range. [3H]ouabain binding inhibition by endobain E was concentration-dependent over a 10-fold range, an effect similar to that found for Na+,K+-ATPase inhibition. The extent of endobain E effect on Na+,K+-ATPase inhibition was much higher (90-100%) than that on [3H]ouabain binding blockade (50%). Findings suggest some type of interaction between endobain E and ouabain inhibitory mechanisms and favour the view that the former behaves as an endogenous ouabain.     

Consequences of mutations to the phosphorylation site of the alpha-subunit of Na, K-ATPase for ATP binding and E1-E2 conformational equilibrium

Expression of Na, K-ATPase in yeast allowed targeting of alpha beta-units with lethal substitutions at the phosphorylation site alpha 1 (D369N) beta 1 and alpha 1 (D369A) beta 1 at the cell surface at the same concentration of alpha-subunit and [3H] ouabain binding sites as for wild type Na, K-ATPase. Phosphorylation and reaction with vanadate were abolished, and the mutations had no Na, K-ATPase or K-phosphatase activity. Binding of [3H]-ATP at equilibrium revealed an intrinsic high affinity of the D369A mutation for ATP (KD = 2.8 nM) that was 39-fold higher than for wild type Na, K-ATPase (KD = 109 nM). The affinities for ADP were unaffected, indicating that the negative charge at residue 369 determines the contribution of the gamma-phosphate to the free energy of ATP binding. Analysis of the K(+)-ATP antagonism showed that the reduction of charge and hydrophobic substitution at Asp369 of the alpha-subunit caused a large shift in conformational equilibrium toward the E2-form. This was accompanied by a large increase in affinity for [3H] ouabain in Mg2+ medium with KD = 4.9 nM for D369A compared to KD = 51 nM for D369N and KD = 133 nM for wild type, and [3H] ouabain binding (KD = 153 nM) to D369A was detectable even in absence of Mg2+. In addition to its function as receptor of the gamma-phosphate of ATP, Asp369 has important short-range catalytic functions in modulating the affinity for ATP and long-range functions in governing the E1-E2 transitions which are coupled to reorientation of cation sites and changes in affinity for digitalis glycosides.     

Inhibition of [3H]norepinephrine uptake in peripheral organs of some mammalian species by ouabain

In order to demonstrate the possible involvement of (Na+ + K+)-ATPase in the high affinity uptake of [3H]-norepinephrine in the sympathetic nerve endings, the effect of ouabain on [3H]norepinephrine uptake in spleen and heart slices of five mammalian species was examined. The ouabain sensitivity of [3H]norepinephrine uptake in the heart slices form various species, as determined by the estimation of IC52, was, in increasing order, lamb (2,3 muM) less than calf (2.5 muM) less than guinea pig (4 muM) less than rabbit (10muM) less than rat (greater than 500 muM). The IC50 values in the spleen slices were: lamb (1 muM) less than calf (3.2 muM) less than rabbit (9.5 muM) less than guinea pig (25 muM) less than rat (greater than 500 muM). The IC50 values for the inhibition of specific [3H]ouabain binding in the microsomal fractions of spleen and heart of the five mammalian species by ouabain were similar to the IC50 values for the inhibition of [3H]norepinephrine uptake by the cardiac glycoside. Since ouabain is known to bind exclusively to (Na+ + K+)-ATPase of a microsomal fraction, these results suggest that the inhibition of [3H]norepinephrine uptake in the sympathetic nerve endings by ouabain is mediated by the inhibition of (Na+ + K+)-ATPase.     

Mechanism of enhanced Na-K-ATPase activity in cortical collecting duct from rats with nephrotic syndrome

The maximal hydrolytic activity of Na-K-ATPase is specifically increased in the cortical collecting duct (CCD) of rats with puromycin-induced nephrotic syndrome (NS). This stimulation is independent of aldosterone and of endogenous ouabain-like substance. To investigate the mechanism responsible for this change, we compared the maximal Na-K-ATPase hydrolytic activity, the ouabain sensitive 86Rb influx, the specific [3H]ouabain binding, and the sensitivity of Na-K-ATPase to ouabain in the CCD of control rats and of rats given an intraperitoneal injection of puromycin 7 d before study. Both Na-K-ATPase activity and ouabain-sensitive 86Rb influx increased two-fold in rats with NS (ATPase activity: 34.1 +/- 2.1 vs. 18.0 +/- 0.7 pmol.mm-1 x min-1 +/- SE, n = 6, P < 0.001; Rb influx: 14.4 +/- 0.7 vs. 7.4 +/- 0.4 peq.min-1 +/- SE, n = 6, P < 0.001) whereas specific [3H]ouabain binding decreased in rats with NS (6.9 +/- 0.7 vs. 9.0 +/- 0.6 fmol.mm-1 +/- SE, n = 6, P < 0.005). Therefore, the maximal turnover rate of Na-K-ATPase increased over twofold in rats with NS (5,053 +/- 361 vs. 2,043 +/- 124 cycles.min-1 +/- SE, n = 6, P < 0.001). Analysis of the curves of inhibition of Na-K-ATPase by ouabain showed the presence of two Na-K-ATPase populations in both control and NS rats: a highly sensitive population (apparent Ki: 1.4 x 10(-6) M and 0.9 x 10(-6) M) and a less sensitive moiety (apparent Ki: 2.6 x 10(-4) M and 1.1 x 10(-4) M). The enhancement of Na-K-ATPase activity observed in the CCD of rats with NS was entirely due to the stimulation of the population of Na-K-ATPase with low ouabain sensitivity. These results suggest that a dysregulation of this subclass of Na-K-ATPase might be the primary cause of sodium retention in this model of nephrotic syndrome.     

Relation of exocytotic release of gamma-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the gamma-aminobutyric acid (GABA) carrier, NNC-711, (1-[(2-diphenylmethylene)amino]oxyethyl)- 1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride, blocks the Ca(2+)-independent release of [3H]GABA from rat brain synaptosomes induced by 50 mM K+ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca(2+)-dependent release of [3H]GABA in the total absence of carrier-mediated release. Reversal of the Na+/Ca2+ exchanger was used to increase the intracellular free Ca2+ concentration ([Ca2+]i) to test whether an increase in [Ca2+]i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca2+]i may rise to values above 400 nM, as a result of Na+/Ca2+ exchange, without inducing release of [3H]GABA, but subsequent K+ depolarization immediately induced [3H]GABA release. Thus, a rise of only a few nanomolar Ca2+ in the cytoplasm induced by 50 mM K+ depolarization, after loading the synaptosomes with Ca2+ by Na+/Ca2+ exchange, induced exocytotic [3H]GABA release, whereas the rise in cytoplasmic [Ca2+] caused by reversal of the Na+/Ca2+ exchanger was insufficient to induce exocytosis, although the value for [Ca2+]i attained was higher than that required for exocytosis induced by K+ depolarization. The voltage-dependent Ca2+ entry due to K+ depolarization, after maximal Ca2+ loading of the synaptosomes by Na+/Ca2+ exchange, and the consequent [3H]GABA release could be blocked by 50 microM verapamil. Although preloading the synaptosomes with Ca2+ by Na+/Ca2+ exchange did not cause [3H]GABA release under any conditions studied, the rise in cytoplasmic [Ca2+] due to Na+/Ca2+ exchange increased the sensitivity to external Ca2+ of the exocytotic release of [3H]GABA induced by subsequent K+ depolarization. Thus, our results show that the vesicular release of [3H]GABA is rather insensitive to bulk cytoplasmic [Ca2+] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca2+ entry through Ca2+ channels near the active zones.     

Importance of intramembrane carboxylic acids for occlusion of K+ ions at equilibrium in renal Na,K-ATPase

Site-directed mutagenesis and assay of Rb+ and Tl+ occlusion in recombinant Na,K-ATPase from yeast were combined to establish structure-function relationships of amino acid side chains involved in high-affinity occlusion of K+ in the E2[2K] form. The wild-type yeast enzyme was capable of occluding 2 Rb+ or Tl+ ions/ouabain binding site or alpha 1 beta 1 unit with high apparent affinity (Kd(Tl+) = 7 +/- 2 microM), like the purified Na,K-ATPase from pig kidney. Mutations of Glu327(Gln,Asp), Asp804(Asn, Glu), Asp808(Asn, Glu) and Glu779(Asp) abolished high-affinity occlusion of Rb+ or Tl+ ions. The substitution of Glu779 for Gln reduced the occlusion capacity to 1 Tl+ ion/alpha 1 beta 1-unit with a 3-fold decrease of the apparent affinity for the ion (Kd(Tl+) = 24 +/- 8 microM). These effects on occlusion were closely correlated to effects of the mutations on K0.5(K+) for K+ displacement of ATP binding. Each of the four carboxylate residues Glu327, Glu779, and Asp804 or Asp808 in transmembrane segments 4, 5, and 6 is therefore essential for high-affinity occlusion of K+ in the E2[2K] form. These residues either may engage directly in cation coordination or they may be important for formation or stability of the occlusion cavity.     

Ca2+ changes and 86Rb efflux activated by hyposmolarity in cerebellar granule neurons

Hyposmotic swelling increased 86Rb release in cultured cerebellar granule neurons (1 day in vitro [DIV]) with a magnitude related to the change in osmolarity. 86Rb release was partially blocked by quinidine, Ba2+, and Cs+ but not by TEA, 4-AP, or Gd3+. 86Rb efflux decreased in Cl(-)-depleted cells or cells treated with DDF or DIDS, suggesting an interconnection between Cl- and K+ fluxes. Swelling induced a substantial increase in [Ca2+]i to which both external and internal sources contribute. However, 86Rb efflux was independent of [Ca2+]0, unaffected by depleting the endoplasmic reticulum (ER) by ionomycin or thapsigargin and insensitive to charybdotoxin, iberiotoxin, and apamin. Swelling-activated 86Rb efflux in differentiated granule neurons after 8 DIV, which express Ca2+-sensitive K+ channels, was not different from that in 1 DIV neurons, nor in time course, net release, Ca2+-dependence, or pharmacological sensitivity. We conclude that the swelling-activated K+ efflux in cerebellar granule neurons is not mediated by Ca2+-sensitive large conductance K+ channels (BK) as in many cell types but resembles that in lymphocytes where it is possibly carried by voltage-gated K+ channels.     

Inhibition of Na+/K+ ATPase under hypertonic conditions in rat mast cells

Ionic fluxes that contribute to changes in membrane potential and variations of pHi (intracellular pH) are not well known in mast cells, although they can be important in the stimulus-secretion coupling. Cellular volume regulation implies changes in the concentration of intracellular ions, such as sodium and potassium and volume changes can be imposed varying the tonicity of the medium. We studied the physiology of sodium and examined the effect of ouabain on [22Na] entry in mast cells in isotonic and hypertonic media. We also recorded changes in membrane potential and pHi using the fluorescent dyes bis-oxonol (Bis-(1,3-diethylthiobarbituric acid) trimethineoxonol) a n d BCECF (2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester) in hypertonic conditions. The results show that [22Na] influx increases four fold in hypertonic solutions and it is mediated mainly by an amiloride-sensitive Na+/H+ exchanger. This transporter is involved in the shrinkage-activated cellular alkalinization and the pHi recovery is accelerated by inhibition of the Na+/K+ ATPase with ouabain in the absence of extracellular calcium. Under hypertonic conditions 22Na influx is apparently not increased by ouabain, while the Na+/K+ ATPase inhibitor clearly increases [22Na] uptake and also induces membrane depolarization in isotonic conditions. All together, these findings suggest that Na+/K+ ATPase is partially inhibited in hypertonic conditions.     

Biomarkers for malignancy in the columnar-lined esophagus

Toads of the genus Bufo are highly resistant to the toxic effects of digitalis glycosides, and the Na+,K(+)-ATPase of all toad tissues studied to date has been relatively insensitive to inhibition by digitalis and related compounds. In studies of brain microsomal preparations from two toad species, Bufo marinus and Bufo viridis, inhibition of ATPase activity and displacement of [3H]ouabain from Na+,K(+)-ATPase occurred over broad ranges of ouabain or bufalin concentrations, consistent with the possibility that more than one Na+,K(+)-ATPase isoform may be present in toad brain. The data could be fitted to one- or two-site models, both of which were consistent with the presence of Na+,K(+)-ATPase activity with high sensitivity to ouabain and bufalin. Ki (concentration capable of producing 50% inhibition of activity) values for ouabain in the one-site model were in the 0.2 to 3.7 microM range, whereas Ki1 values in the two-site model ranged from 0.085 to 0.85 microM, indicating that brain ATPase was at least three orders of magnitude more sensitive to ouabain than B. marinus bladder ATPase (Ki = 5940 microM). Ouabain was also an effective inhibitor of 86Rb+ uptake in B. marinus brain tissue slices (Ki = 3.1 microM in the one-site model; Ki1 = 0.03 microM in the two-site model). However, the relative contribution of the high ouabain-sensitivity site to the total activity was 17% in the transport assay as compared with 63% in the Na+,K(+)-ATPase enzymatic assay. We conclude that a highly ouabain-sensitive Na+,K(+)-ATPase activity is present and functional in toad brain but that its function may be partially inhibited in vivo.     

Small changes in extracellular sodium influence myogenic tone in rabbit facial vein by changing its sensitivity to calcium

Extracellular Na+ concentration ([Na+]e) significantly effects the regulation of myogenic tone in isolated blood vessels. We examined the effect of small changes in [Na+]e on simultaneous changes in stretch-activated myogenic tone in rabbit facial vein and 45Ca2+ unidirectional influx and net uptake. Decreasing [Na+]e from 150 to 120 mmol/l augmented myogenic tone (control: 3.15 +/- 0.27 mN, n = 22) by 89 +/- 29%, while raising [Na+]e to 165 mmol/l attenuated myogenic tone to 80 +/- 2% of control. Changes in myogenic tone induced by alterations in [Na+]e were not accompanied by proportional changes in 45Ca2+ net uptake. 45Ca2+ unidirectional influx per unit of wall force (10.2 +/- 1.0 pmol/mg per mN force, n = 22, control) was decreased to 6.1 +/- 0.6 pmol/mg per mN (n = 20, P < 0.05) and increased to 21.0 +/- 2.5 pmol/mg per mN (n = 14, P < 0.05) when [Na+]e was 120 or 165 mmol/l, respectively, suggesting that decreasing [Na+]e is related to an increased sensitivity to calcium. We conclude that, in the rabbit facial vein, the sensitivity of myogenic tone to changes in [Na+]e may reflect changes in the sensitivity of smooth muscle to Ca2+ through a change in mechanoreceptor sensitivity.     

Calcium-activated potassium conductances on cultured nontransformed dog pancreatic duct epithelial cells

Pancreatic duct epithelial cells (PDECs) mediate the pancreatic secretion of fluid and electrolytes. Membrane K+ channels on these cells regulate intracellular K+ concentration; in combination with the Na+/H+ antiport and Na+,K+ adenosine triphosphatase (ATPase), they may also mediate serosal H+ secretion, balancing luminal HCO3- secretion. We describe the K+ conductances on well-differentiated and functional nontransformed cultured dog PDECs. Through 86Rb+ efflux studies, we demonstrated Ca(2+)-activated K+ channels that were stimulated by A23187, thapsigargin, and 1-ethyl-2-benzimidazolinone, but not forskolin. These conductances also were localized on the basolateral membrane because 86Rb+ efflux was directed toward the serosal compartment. Of the K+ channel blockers, BaCl2, charybdotoxin, clotrimazole, and quinidine, but not 4-aminopyridine, apamin, tetraethylammonium, or iberiotoxin, inhibited 86Rb+ efflux. This efflux was not inhibited by amiloride, ouabain, and bumetanide, inhibitors of the Na+/H+ antiport, the Na+,K(+)-ATPase pump, and the Na+,K+,2Cl- cotransporter, respectively. When apically permeabilized PDEC monolayers were mounted in Ussing chambers with a luminal-to-serosal K+ gradient, A23187 and 1-ethyl-2-benzimidazolinone stimulated a charybdotoxin-sensitive short-circuit current (Isc) increase. Characterization of K+ channels on these cultured PDECs, along with previous identification of Cl- channels (1), further supports the importance of these cells as models for pancreatic duct secretion.     

Asp804 and Asp808 in the transmembrane domain of the Na,K-ATPase alpha subunit are cation coordinating residues

The functional roles of Asp804 and Asp808, located in the sixth transmembrane segment of the Na,K-ATPase alpha subunit, were examined. Nonconservative replacement of these residues yielded enzymes unable to support cell viability. Only the conservative substitution, Ala808 --> Glu, was able to maintain the essential cation gradients (Van Huysse, J. W., Kuntzweiler, T. A., and Lingrel, J. B (1996) FEBS Lett. 389, 179-185). Asp804 and Asp808 were replaced by Ala, Asn, and Glu in the sheep alpha1 subunit and expressed in a mouse cell line where [3H]ouabain binding was utilized to probe the exogenous proteins. All of the heterologous proteins were targeted into the plasma membrane, bound ouabain and nucleotides, and adopted E1Na, E1ATP, and E2P conformations. K+ competition of ouabain binding to sheep alpha1 and Asp808 --> Glu enzymes displayed IC50 values of 4.11 mM (nHill = 1.4) and 23.8 mM (nHill = 1.6), respectively. All other substituted proteins lacked this K+-ouabain antagonism, e.g. 150 mM KCl did not inhibit ouabain binding. Na+ antagonized ouabain binding to all the expressed isoforms, however, the proteins carrying nonconservative substitutions displayed reduced Hill coefficients (nHill 相似文献   

Effects of glucose deprivation, chemical hypoxia, and simulated ischemia on Na+ homeostasis in rat spinal cord astrocytes

A steep inwardly directed Na+ gradient is essential for glial functions such as glutamate reuptake and regulation of intracellular ion concentrations. We investigated the effects of glucose deprivation, chemical hypoxia, and simulated ischemia on intracellular Na+ concentration ([Na+]i) in cultured spinal cord astrocytes using fluorescence ratio imaging with sodium-binding benzofuran isophthalate (SBFI) AM. Glucose removal or chemical hypoxia (induced by 10 mM NaN3) for 60 min increased [Na+]i from a baseline of 8.3 to 11 mM. Combined glycolytic and respiratory blockage by NaN3 and 0 glucose saline caused [Na+]i to increase by 20 mM, similar to the [Na+]i increases elicited by blocking the Na+/K+-ATPase with ouabain. Recovery from large [Na+]i increases (>15 mM) induced by the glutamatergic agonist kainate was attenuated during glucose deprivation or NaN3 application and was blocked in NaN3 and 0 glucose. To mimic in vivo ischemia, we exposed astrocytes to NaN3 and 0 glucose saline containing L-lactate and glutamate with increased [K+] and decreased [Na+], [Ca2+], and pH. This induced an [Na+]i decrease followed by an [Na+]i rise and a further [Na+]i increase after reperfusion with standard saline. Similar multiphasic [Na+]i changes were observed after NaN3 and 0 glucose saline with only reduced [Na+]e. Our results suggest that the ability to maintain a low [Na+]i enables spinal cord astrocytes to continue uptake of K+ and/or glutamate at the onset of energy failure. With prolonged energy failure, however, astrocytic [Na+]i rises; with loss of their steep transmembrane Na+ gradient, astrocytes may aggravate metabolic insults by carrier reversal and release of acid, K+, and/or glutamate into the extracellular space.