86Rb Uptake Research Articles

The effect of long-term treatment with spironolactone on sodium pump abnormalities in the red blood cells of patients with Bartter's syndrome.

To elucidate the possible sodium transport alterations across the cell membranes in Bartter's syndrome and their influencing by spironolactone treatment Na+-K+-ATPase activity was studied by means of radioactive 86Rubidium influx into red blood cells (RBC) of patients with Bartter's syndrome prior to and after a long-term spironolactone administration. As compared with the control subjects and patients with primary aldosteronism the patients with Bartter's syndrome had a more than 5 times higher 86Rb uptake by the RBC, especially in the ouabain-sensitive component. A long-term spironolactone treatment led to the decrease of this high influx. Serum of patients with Bartter's syndrome incubated with healthy RBC distinctly increased their 86Rb influx. The increase nevertheless did not reach the values in the RBC of untreated patients with Bartter's syndrome. Even if our results do not allow to explain fully the mechanism responsible for the Na+-K+-ATPase changes in the RBC of these patients, analysis of the studied parameters demonstrated that none of the known humoral factors as aldosterone, renin, prostaglandins, or changes of the serum potassium were responsible for these abnormalities. The changes of sodium transport in RBC of patients with Bartter's syndrome could be a part of a more general disturbance of the transport mechanism and could significantly participate in the pathogenesis of this disease.

The Ste20 Kinases Ste20-related Proline-Alanine-rich Kinase and Oxidative-stress Response 1 Regulate NKCC1 Function in Sensory Neurons

NKCC1 is highly expressed in dorsal root ganglion neurons, where it is involved in gating sensory information. In a recent study, it was shown that peripheral nerve injury results in increased NKCC1 activity, not due to an increase in cotransporter expression, but to increased phosphorylation of the cotransporter (Pieraut, S., Matha, V., Sar, C., Hubert, T., Méchaly, I., Hilaire, C., Mersel, M., Delpire, E., Valmier, J., and Scamps, F. (2007) J. Neurosci. 27, 6751-6759). Our laboratory has also identified two Ste20-like kinases that bind and phosphorylate NKCC1: Ste20-related proline-alanine-rich kinase (SPAK) and oxidative-stress response 1 (OSR1). In this study, we show that both kinases are expressed at similar expression levels in spinal cord and dorsal root ganglion neurons, and that both kinases participate equally in the regulation of NKCC1. Using a novel fluorescence method to assay NKCC1 activity in single cells, we show a 50% reduction in NKCC1 activity in DRG neurons isolated from SPAK knockout mice, indicating that another kinase, e.g. OSR1, is present to phosphorylate and activate the cotransporter. Using a nociceptive dorsal root ganglion sensory neuronal cell line, which expresses the same cation-chloride cotransporters and kinases as native DRG neurons, and gene silencing via short hairpin RNA, we demonstrate a direct relationship between kinase expression and cotransporter activity. We show that inactivation of either kinase significantly affects NKCC1 activity, whereas inactivation of both kinases results in an additive effect. In summary, our study demonstrates redundancy of kinases in the regulation of NKCC1 in dorsal root ganglion neurons.

Analysis of exercise‐induced Na+–K+ exchange in rat skeletal muscle in vivo

We aimed to quantify the Na(+)-K(+) exchange occurring during exercise in rat skeletal muscle in vivo. Intracellular Na(+) and K(+) content, Na(+) permeability ((22)Na(+) influx), Na(+)-K(+) pump activity (ouabain-sensitive (86)Rb(+) uptake) and Na(+)-K(+) pump alpha(2) subunit content ([(3)H]ouabain binding) were measured. Six-week-old rats rested (control animals) or performed intermittent running for 10-60 min and were then killed or were killed at 15 or 90 min following 60 min exercise. In the soleus muscle, intracellular Na(+) was 80% higher than in control rats after 60 min exercise, was still elevated (38%) after 15 min rest and returned to control levels after 90 min rest. Intracellular K(+) showed corresponding decreases after 15-60 min exercise, returning to control levels 90 min postexercise. Exercise induced little change in Na(+) and K(+) in the extensor digitorum longus muscle (EDL). In soleus, the exercise-induced rise in Na(+) and reduction in K(+) were augmented by pretreatment with ouabain or by reducing the content of muscular Na(+)-K(+) pumps by prior K(+) depletion of the animals. Fifteen minutes after 60 min exercise, ouabain-sensitive (86)Rb(+) uptake in the soleus was increased by 30% but was unchanged in EDL, and there was no effect of exercise on [(3)H]ouabain binding measured in vitro or in vivo in either muscle. In conclusion, in the soleus, in vivo exercise induces a rise in intracellular Na(+), which reflects the excitation-induced increase in Na(+) influx and leads to augmented Na(+)-K(+) pump activity without apparent change in Na(+)-K(+) pump capacity.

Structural and Functional Interactions of KCl Cotransport Proteins KCC1 and KCC3 in Sickle and Normal Erythrocyte Membranes

The KCl cotransporter (KCC) mediates volume reduction in normal reticulocytes and exaggerated KCC activity in sickle red blood cells (SS RBC) (Joiner et al, Blood 109:1728, 2007) contributes to pathological dehydration that potentiates sickling. Three separate genes (KCC1, KCC3, KCC4) are expressed in RBC (Crable et al, Exp. Hem. 33:624, 2005). KCC1 and KCC3 proteins have been shown to interact in ex vivo expression systems (Simard et al, JBC 282(25):18083, 2007), and co-expression of an N-terminal truncation of KCC1 reduces KCC activity mediated by full-length KCC1 or KCC3 (Casula et al. JBC 276:41870, 2001), suggesting functional interaction. We show here via western blot analysis that SS RBC membranes contain more KCC1 protein (relative to KCC3) than AA RBC, independent of the reticulocytosis of sickle blood. Immunoprecipitation of solubilized SS RBC membranes with KCC3-specific antibody yielded a band at 125 kD on SDS PAGE which contained KCC1, as identified by western blotting with KCC1-specific antibody and by TOF mass spectroscopy. The effect of co-expression of KCC1 and KCC3 on KCC activity was assessed by measuring NEM-stimulated, Cl-dependent, (ouabain + bumetanide)-insensitive Rb uptake in HEK 293 cells. The Flip-In T-rex HEK 293 cell line (Invitrogen) containing a tetracycline-response promoter was transfected with a pcDNA5a plasmid containing KCC3a cDNA. Recombination of the plasmid with the integrated tet-promoter construct inserts the KCC3a gene under control of a tetracycline-responsive promoter. These cells were subsequently transduced with a retroviral vector (SF-91. Hildinger et at, Gene Ther . 5:1575, 1998) containing KCC1 cDNA linked to a GFP cassette. Control cells contained SF-91 vector lacking KCC1. Cells were selected for GFP expression and grown in the absence (un-induced, no KCC3a expression) or presence of tetracycline (induced, KCC3a expression). From this binary matrix, four types of cells were obtained: Cells with no additional KCC expression, representing endogenous KCC activity; cells with only KCC1 or KCC3a expression; cells with both KCC1 and KCC3a expression. Western blots indicated similar KCC1 expression in cells with KCC1 only and [KCC1 + KCC3] and similar KCC3 expression in cells with KCC3 only and [KCC1 + KCC3]. Thus, the expression of neither isoform was affected by the presence of the other. KCC activity in cells overexpressing KCC1 only was similar to endogenous activity in HEK 293 cells; i.e., transport activity of KCC1 alone was minimal. Cells overexpressing KCC3 only had a 5-fold increase in KCC activity over endogenous levels. When KCC1 was co-expressed with KCC3 in [KCC1 + KCC3] cells, an additional 50% increase in KCC activity was observed (p < 0.05 by paired t-test, N=4), despite similar levels of KCC3 expression by western blot analysis. This synergistic effect was dependent on the cytoplasmic N-terminus of KCC1, as it was not seen when the first 39 amino acids of KCC1 were removed. Interestingly, removal of the entire cytoplasmic N-terminal domain (117 aa) produced an inhibitory effect when co-expressed with KCC3a in HEK cells, as previously reported in Xenopus oocytes (Casula et al.). These data indicate that KCC1 and KCC3 interact structurally and functionally in RBC membranes, and provide another potential mechanism for regulation of KCC activity via multimeric associations between KCC isoforms. Thus, KCC activity could be modulated not only by transcriptional mechanisms and post-translational modification (phosphorylation), but also by altering the ratios of KCC isoforms or the kinetics of their association. We speculate that higher levels of KCC1 protein relative to KCC3 in SS RBC membranes could account for higher KCC activity in these cells relative to AA RBC.

Diverse Functional Consequences of Mutations in the Na+/K+-ATPase α2-Subunit Causing Familial Hemiplegic Migraine Type 2

Mutations in ATP1A2, the gene coding for the Na(+)/K(+)-ATPase alpha(2)-subunit, are associated with both familial hemiplegic migraine and sporadic cases of hemiplegic migraine. In this study, we examined the functional properties of 11 ATP1A2 mutations associated with familial or sporadic hemiplegic migraine, including missense mutations (T263M, T376M, R383H, A606T, R763H, M829R, R834Q, R937P, and X1021R), a deletion mutant (del(K935-S940)ins(I)), and a frameshift mutation (S966fs). According to the Na(+)/K(+)-ATPase crystal structure, a subset of the mutated residues (Ala(606), Arg(763), Met(829), and Arg(834)) is involved in important interdomain H-bond networks, and the C terminus of the enzyme, which is elongated by the X1021R mutation, has been implicated in voltage dependence and formation of a third Na(+)-binding site. Upon heterologous expression in Xenopus oocytes, the analysis of electrogenic transport properties, Rb(+) uptake, and protein expression revealed pronounced and markedly diverse functional alterations in all ATP1A2 mutants. Abnormalities included a complete loss of function (T376M), impaired plasma membrane expression (del(K935-S940)ins(I) and S966fs), and altered apparent affinities for extracellular cations or reduced enzyme turnover (R383H, A606T, R763H, R834Q, and X1021R). In addition, changes in the voltage dependence of pump currents and the increased rate constants of the voltage jump-induced redistribution between E(1)P and E(2)P states were observed. Thus, mutations that disrupt distinct interdomain H-bond patterns can cause abnormal conformational flexibility and exert long range consequences on apparent cation affinities or voltage dependence. Of interest, the X1021R mutation severely impaired voltage dependence and kinetics of Na(+)-translocating partial reactions, corroborating the critical role of the C terminus of Na(+)/K(+)-ATPase in these processes.

Modulation of insulin release by adenosine A1 receptor agonists and antagonists in INS‐1 cells: The possible contribution of 86Rb+ efflux and 45Ca2+ uptake

Due to the lack of specific agonists and antagonists the role of adenosine receptor subtypes with respect to their effect on the insulin secretory system is not well investigated. The A1 receptor may be linked to different 2nd messenger systems, i.e. cAMP, K+- and 45Ca2+ channel activity. Partial A1 receptor agonists are going to be developed in order to improve diabetes (increase in insulin sensitivity, lowering of FFA and triglycerides). In this study newly synthesized selective A1 receptor agonists and antagonists were investigated thereby integrating three parameters, insulin release (RIA), 45Ca2+ uptake and 86Rb+ efflux (surrogate for K+ efflux) of INS-1 cells, an insulin secretory cell line. The presence of A1-receptors was demonstrated by Western blotting. The receptor nonselective adenosine analogue NECA (5-N-ethylcarboxyamidoadenosine) at high concentration (10 microM) had no effect on insulin release and 45Ca2+ uptake which could be interpreted as the sum of effects mediated by mutual antagonistic adenosine receptor subtypes. However, an inhibitory effect mediated by A1 receptor agonism was detected at 10 nM NECA and could be confirmed by adding the A1 receptor antagonist PSB-36 (1-butyl-8-(3-noradamantyl)-3-(3-hydroxy-propyl)xanthine). NECA inhibited 86Rb+ efflux which, however, did not fit with the simultaneous inhibition of insulin secretion. The selective A1 receptor agonist CHA (N6-cyclohexyladenosine) inhibited insulin release; the simultaneously increased Ca2+ uptake (nifedipine dependent) and inhibition of 86Rb+ efflux did not fit the insulin release data. The CHA effect (even the maximum effect at 50 microM) can be increased by 10 microM NECA indicating that CHA and NECA have nonspecific and physiologically non-relevant effects on 86Rb+ efflux in addition to their A1-receptor interaction. Since PSB-36 did not influence the NECA-induced inhibition of 86Rb+ efflux, the NECA effect is not mediated by potassium channel-linked A1 receptors. The nonselective adenosine receptor antagonist caffeine increased insulin release which was reversed by CHA as expected when hypothesizing that both act via A1 receptors in this case. In conclusion, stimulation of A1 receptors by receptor selective and nonselective compounds reduced insulin release which is not coupled to opening of potassium channels (86Rb+ efflux experiments) or inhibition of calcium channels (45Ca2+ uptake experiments). It may be expected that of all pleiotropic 2nd messengers, the cAMP system (not tested here) is predominant for A1 receptor effects and the channel systems (K+ and Ca2+) are of minor importance and do not contribute to insulin release though being coupled to the receptor in other tissues.

AtCCX3 Is an Arabidopsis Endomembrane H+-Dependent K+ Transporter

The Arabidopsis (Arabidopsis thaliana) cation calcium exchangers (CCXs) were recently identified as a subfamily of cation transporters; however, no plant CCXs have been functionally characterized. Here, we show that Arabidopsis AtCCX3 (At3g14070) and AtCCX4 (At1g54115) can suppress yeast mutants defective in Na(+), K(+), and Mn(2+) transport. We also report high-capacity uptake of (86)Rb(+) in tonoplast-enriched vesicles from yeast expressing AtCCX3. Cation competition studies showed inhibition of (86)Rb(+) uptake in AtCCX3 cells by excess Na(+), K(+), and Mn(2+). Functional epitope-tagged AtCCX3 fusion proteins were localized to endomembranes in plants and yeast. In Arabidopsis, AtCCX3 is primarily expressed in flowers, while AtCCX4 is expressed throughout the plant. Quantitative polymerase chain reaction showed that expression of AtCCX3 increased in plants treated with NaCl, KCl, and MnCl(2). Insertional mutant lines of AtCCX3 and AtCCX4 displayed no apparent growth defects; however, overexpression of AtCCX3 caused increased Na(+) accumulation and increased (86)Rb(+) transport. Uptake of (86)Rb(+) increased in tonoplast-enriched membranes isolated from Arabidopsis lines expressing CCX3 driven by the cauliflower mosaic virus 35S promoter. Overexpression of AtCCX3 in tobacco (Nicotiana tabacum) produced lesions in the leaves, stunted growth, and resulted in the accumulation of higher levels of numerous cations. In summary, these findings suggest that AtCCX3 is an endomembrane-localized H(+)-dependent K(+) transporter with apparent Na(+) and Mn(2+) transport properties distinct from those of previously characterized plant transporters.

Influence of Silicon on Sunflower Cultivars under Drought Stress, II: Essential and Nonessential Element Uptake determined by Polarized Energy Dispersive X‐ray Fluorescence

: There is no information regarding genotypic variation in essential and nonessential nutrient accumulation of sunflower grown under drought stress with the presence or absence of supplemental silicon (Si) despite the role of this element in improving growth of some cultivars under drought conditions. Accumulation of elements in sunflower cultivars might be important for the genetic improvement of the crop's response to drought. An experiment under controlled conditions was carried out to study the genotypic response of 12 sunflower (Helianthus annuus L.) cultivars to drought and Si and the relationship to the uptake of elements [phosphorus (P), potassium (K), sulfur (S), calcium (Ca), magnesium (Mg), iron (Fe), copper (Cu), zinc (Zn), manganese (Mn), chloride (Cl), molybdenum (Mo), Si, sodium (Na), vanadium (V), aluminum (Al), strontium (Sr), rubidium (Rb), titanium (Ti), chromium (Cr), nickel (Ni), bromine (Br), and barium (Ba)]. This was determined by polarized energy‐dispersive X‐ray fluorescence (PEDXRF). It was observed that uptake of nutrient and nonessential elements by sunflower cultivars were differentiated in response to applied Si and drought stress. Drought stress decreased mineral uptake of all the cultivars, and generally, application of Si under drought stress significantly improved Si, K, S, Mg, Fe, Cu, Mn, Na, Cl, V, Al, Sr, Rb, Ti, Cr, and Ba uptake whereas Zn, Mo, Ni, and Br uptake were not affected.

Li fluxes reveal presence of Na‐Cl Cotransport (NCC) in Human Lens Epithelial Cells (LECs)

Na‐K‐2Cl cotransport (NKCC) moves RbCl/KCl+NaCl/LiCl and, in FHL124 LECs, is not inhibited by hypotonicity. Here, we studied Li fluxes through NKCC in hyposmotic high K media and Rb fluxes in hyposmotic high Na to understand this effect. Li and Rb uptake were measured by atomic absorption spectrophotometry to determine LiKCC or NRbCC through bumetanide‐sensitive (BS) and Cl‐dependent fluxes in Na/K or Na‐free/K‐free (N‐methyl‐D‐glucamine) and Cl or Cl‐free (sulfamate or nitrate) media ± ouabain (O) or [O+B], ± thiazides, at varying Rb, Li or Cl molar fractions (MF). Main findings are: Li influx in isosmotic (300 mOsM) K was 1/3 of Rb influx in Na, and was mainly mediated by a Cl‐dependent Li flux (LiKCC). In 200 mOsM high K, LiKCC was abolished, whereas in high Na, NRbCC remained active. LiKCC and BS‐Li influx (BS‐LiKCC) showed bell‐shape curves for 0.1–1 Li MF, maxing at ∼0.6 MF. BS‐LiKCC was ¼ of LiKCC. The difference, i.e. the B‐insensitive/Cl‐dependent‐Li influx, saturated with Li and Cl MFs. Kms for Li were 11 with and 7–8 mM without external K, respectively; and ∼40 mM for Cl. Neither furosemide (&lt;100 μM) nor thiazide derivatives inhibited LiCC. RTPCR, Western blots and immunochemistry revealed NCC RNA and protein, with NCC‐specific antibodies, besides the expected NKCC1. Thus, a K‐independent/Cl‐dependent Li flux (LiCC) revealed the presence of NCC in FHL124 LECs.

Nanomolar concentrations of ouabain stimulate Na‐K ATPase through sodium hydrogen exchanger‐1 (NHE1) dependent mechanism.

Recent investigations have shown increased Na‐H exchanger activity and plasma levels of endogenous cardiotonic glycosides, such as ouabain in spontaneously hypertensive rats. At high concentrations, ouabain inhibits pump activity, and at lower concentrations, it increases pump activity and induces cell proliferation. Stimulation of NHE1 and Na‐K ATPase activity involve protein kinase B (AKT), suggesting that both NHE1 and Na‐K ATPase are mediated by the same proteins. We hypothesize that the activity of NHE1 and Na‐K ATPase are linked. In the present study we aim to 1) determine the role of activation of NHE1 in ouabain‐sensitive stimulation of Na‐K ATPase, 2) determine if NHE1 and Na‐K ATPase association increases when cells were treated with NH4Cl or ouabain in various kidney proximal tubule cell lines (OK, HK2, HK5, and HK11). Our data suggests that NH4Cl stimulation of NHE1 increases ouabain sensitive 86Rb uptake in all cell lines examined.Treatment with ethylisopropylamiloride (EIPA) a NHE1 specific inhibitor decreased Na‐K ATPase activity in HK5 and HK11 cells and prevented nanomolar ouabain‐stimulated Na‐K ATPase activity in OK cells. Immunoblot analysis indicated that NH4Cl increased membrane expression of NHE1 and Na‐K ATPase in OK and HK11 cells. Surprisingly, treatment with EIPA alone increased membrane expression of NHE1 and Na‐K ATPase. The reason for increased presence of these proteins in the membrane is unknown, however, tyrosine phosphorylation of the catalytic subunit of Na‐K ATPase, believed to be an indication of Na‐K ATPase activation, is decreased under these same conditions. These results suggest that activation of NHE1 is critical for ouabain stimulated increase in Na‐K ATPase activity, and that upon activation, NHE1 and Na‐K ATPase associate, perhaps to form a signalosome.

CT-based attenuation correction in 82Rb-myocardial perfusion PET–CT: incidence of misalignment and effect on regional tracer distribution

Misalignment of low-dose-CT used for attenuation correction (AC) may cause artifacts in cardiac-PET-CT. The aim was to evaluate incidence and severity of misalignment and its quantitative effects on regional myocardial (82)Rb-distribution. Rest/dipyridamole (82)Rb-perfusion-PET-CT studies of 92 consecutive patients were analyzed for misalignment. Two different scanning protocols were employed: the first 57 patients had separate CTs for rest and stress PET. The following 35 patients had one CT at rest, used for AC of rest and stress PET. Misalignment was visually scored on a five-point scale (0 = no, 1 = minimal, 2 = mild, 3 = moderate, and 4 = severe). In five representative patients with normal perfusion and low probability of disease, 95 polarmaps were created by shifting CT vs PET prior to reconstruction of attenuation-corrected data sets using dedicated software (three dimensions of space; magnitude of shifts, 5, 10, 14 mm). PET/CT -misalignment was detected in 60% of rest and 67% of stress studies. Alignment for rest was better than that for stress (0.7 +/- 0.7 vs 1.0 +/- 0.9, P = 0.03). Comparison of the two protocols revealed no effect on the alignment of the stress study (1.0 +/- 0.9 vs 1.0 +/- 0.9, P = 0.9). Quantitatively, the largest individual effect of any artificial misalignment was a 25% reduction of relative (82)Rb uptake. With a shift of 1 cm, the largest effect in an individual was a 19% decrease. Anterior wall was most frequently influenced by misalignment, but changes of uptake also occurred in all other segments. Misalignment between CT and PET in cardiac-PET-CT influences regional tracer distribution in multiple segments. Repeated CT imaging after dipyridamole does not improve alignment. These results emphasize the need for strategies to improve coregistration in clinical imaging protocols.

Palytoxin Acts on Na+,K+-ATPase but not Nongastric H+,K+-ATPase

Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on nongastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase alpha(2)- and Na,K-ATPase beta(2)-subunits; Bufo Na,K-ATPase alpha(1)- and Na,K-ATPase beta(2)-subunits; and Bufo Na,K-ATPase beta(2)-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 microM ouabain. Functional expression was confirmed by measuring (86)Rb uptake. PTX (5 nM: ) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo beta(2)-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase alpha(1)-subunit and Na,K-ATPase beta(1)-subunit; rat Na,K-ATPase alpha(2)-subunit and Na,K-ATPase beta(2)-subunit; and rat Na,K-ATPase beta(1)- or Na,K-ATPase beta(2)-subunit alone. Measurement of increases in (86)Rb uptake confirmed that both rat Na,K and H,K pumps were functional in HeLa cells expressing rat colonic HKalpha(1)/NKbeta(1) and NKalpha(2)/NKbeta(2). Whole-cell patch-clamp measurements in HeLa cells expressing rat colonic HKalpha(1)/NKbeta(1) exposed to 100 nM PTX showed no significant increase of membrane current, and there was no membrane conductance increase in HeLa cells transfected with rat NKbeta(1)- or rat NKbeta(2)-subunit alone. However, in HeLa cells expressing rat NKalpha(2)/NKbeta(2), outward current was observed after pump activation by 20 mM K(+) and a large membrane conductance increase occurred after 100 nM PTX. We conclude that nongastric H,K-ATPases are not sensitive to PTX when expressed in these cells, whereas PTX does act on Na,K-ATPase.

Presence of a functional TSH receptor on human erythrocytes

The presence of thyrotropin receptors (TSHR) has been reported in some extrathyroidal tissues but its physio-pathological role still remains unclear. TSH (seems to) affects the human erythrocytes Na +/K +-ATPase in vitro, however receptors on erythrocytes have not yet been described. In this work the effect of recombinant human TSH (rhTSH) on sites number and activity of erythrocyte Na +/K +-ATPase was investigated in a group of thyroidectomized patient enrolled for rhTSH test. As detected by 3H-ouabain binding, rhTSH administration induced a significant increase in the number of sites ( p = 0.005) and in the Kd ( p = 0.006) of Na +/K +-ATPase. rhTSH did not induce significant difference in Na +/K +-ATPase activity measured by 86Rb uptake. 125I-TSH binding studies and Western blotting data showed the existence of TSHR in the erythrocytes of healthy donors. In conclusion The TSH action on Na +/K +-ATPase of human erythrocytes can be explained by the presence of TSHR

Purinergic agonists stimulate lens Na-K-ATPase-mediated transport via a Src tyrosine kinase-dependent pathway

The Na-K-ATPase is vital for maintenance of lens transparency. Past studies using intact lens suggested the involvement of tyrosine kinases in short-term regulation of Na-K-ATPase. Furthermore, in vitro phosphorylation of a lens epithelial membrane preparation by Src family kinases (SFKs), a family of nonreceptor tyrosine kinases, resulted in modification of Na-K-ATPase activity. Here, the effect of purinergic agonists, ATP and UTP, on Na-K-ATPase function and SFK activation was examined in the rabbit lens. Na-K-ATPase function was examined using two different approaches, measurement of ouabain-sensitive potassium ((86)Rb) uptake by the intact lens, and Na-K-ATPase activity in lens epithelial homogenates. ATP and UTP caused a significant increase in ouabain-sensitive potassium ((86)Rb) uptake. Na-K-ATPase activity was increased in the epithelium of lenses pretreated with ATP. Lenses treated with ATP or UTP displayed activation of SFKs as evidenced by increased Western blot band density of active SFK (phosphorylated at the active loop Y416) and decreased band density of inactive SFKs (phosphorylated at the COOH terminal). A single PY416-Src immunoreactive band at approximately 60 kDa was observed, suggesting not all Src family members are activated. Immunoprecipitation studies showed that band density of active Src, and to a lesser extent active Fyn, was significantly increased, while active Yes did not change. Preincubation of the lenses with SFK inhibitor PP2 abolished the ATP-induced increase in ouabain-sensitive potassium ((86)Rb) uptake. The results suggest selective activation of Src and/or Fyn is part of a signaling mechanism initiated by purinergic agonists that increases Na-K-ATPase-mediated transport in the organ-cultured lens.

Rubidium chloride tolerant callus cultures of rice (Oryza sativa L.) accumulate more potassium and cross tolerate to other salts

Callus cultures from salt tolerant (CSR-10) and susceptible (Swarnadhan) varieties of Oryza sativa L. were established in Murashige and Skoog's (MS) medium containing lethal concentrations (50 mM) of rubidium chloride (RbCl) as a selective agent. While 95-100% cells were viable in callus cultures grown without RbCl, viability was 75% in 50 mM RbCl selected cultures. Growth of RbCl selected calli in presence of salt was comparable to that of callus grown without it. Cells tolerant to RbCl showed more vacuoles and accumulated more K(+) in comparison with their corresponding controls. Suspension cultures were established and uptake of (86)Rb(+) was measured at 10 and 20 min intervals, which revealed a linear relationship between the absorption of K(+) and time. Callus cultures (560-day-old) tolerant to 50 mM RbCl regenerated shoots with 35-40% frequencies in both the varieties, but the same age-old callus grown in the medium devoid of RbCl did not show any organogenesis. Callus cultures that are tolerant to 50 mM RbCl when exposed to 25 mM LiCl, 50 mM NaCl, 50 mM KCl and 25 mM CsCl also exhibited cross tolerance in both the varieties. This is the first time that a callus line of rice resistant to RbCl was raised and shown to accumulate a major cation K(+ )and also an increased influx of it.

Increase in the toxic effects of Tl+ on isolated rat liver mitochondria in the presence of nonactin

The effects of Tl(+) ions on isolated rat liver mitochondria were studied in the presence of nonactin, a cyclic ionophore. Nonenergized rat liver mitochondria were increasingly swollen at an elevated concentration of Tl(+) in the 160 mOsm medium containing 0-150 mM sucrose and 0-75 mM TlNO(3) or 0-50 mM Tl acetate. On the contrary, mitochondria in experiments with nonactin were contracted in the medium with 5-25 mM Tl(+) and were swollen only in the medium with 50-75 mM TlNO(3) or 50 mM Tl acetate. State 4 respiration along with swelling of succinate-energized mitochondria followed contraction after their deenergization was further enhanced at increasing concentration of Tl acetate in a medium containing nonactin. Regardless of the presence of nonactin, State 3 and 2,4-dinitrophenol (DNP)-stimulated respiration and the monoamine oxidase (MAO) activity were not affected in the medium with 0-25 mM Tl acetate and sucrose. DNP-stimulated respiration decreased and the MAO activity somewhat increased in the medium containing 50 mM Tl acetate and nonactin. Uptake of (86)Rb(+) by energized mitochondria in the presence of valinomycin was considerably decreased when Tl(+) and nonactin were simultaneously present in the medium. An increase of the toxic effect of Tl(+) on rat liver mitochondria in the presence of nonactin is accounted for by disruption of mitochondria due to their more extensive swelling and uncoupling of mitochondria, resulting in the stimulation of State 4 and depletion of their energy store.

The Na+ transporter AtHKT1;1 controls retrieval of Na+ from the xylem in Arabidopsis

HKT-type transporters appear to play key roles in Na(+) accumulation and salt sensitivity in plants. In Arabidopsis HKT1;1 has been proposed to influx Na(+) into roots, recirculate Na(+) in the phloem and control root : shoot allocation of Na(+). We tested these hypotheses using (22)Na(+) flux measurements and ion accumulation assays in an hkt1;1 mutant and demonstrated that AtHKT1;1 contributes to the control of both root accumulation of Na(+) and retrieval of Na(+) from the xylem, but is not involved in root influx or recirculation in the phloem. Mathematical modelling indicated that the effects of the hkt1;1 mutation on root accumulation and xylem retrieval were independent. Although AtHKT1;1 has been implicated in regulation of K(+) transport and the hkt1;1 mutant showed altered net K(+) accumulation, (86)Rb(+) uptake was unaffected by the hkt1;1 mutation. The hkt1;1 mutation has been shown previously to rescue growth of the sos1 mutant on low K(+); however, HKT1;1 knockout did not alter K(+) or (86)Rb(+) accumulation in sos1.

Parathyroid Hormone Regulates Na‐K ATPase through ERK‐dependent cPLA2 phosphorylation

We have demonstrated that PTH regulates Na-K ATPase (Na-K) through ERK-dependent PKC alpha activation that results in translocation of PKC alpha to the plasma membrane and association with Na-K. Based on previous data showing an inhibitory effect of PLA2 products on Na-K activity, we hypothesized that PTH-stimulated ERK activates PKC alpha through a PLA2 dependent mechanism. To address this hypothesis, we examined the effect of PTH on PLA2 and the effect of cPLA2 products on activation of PKC alpha in opossum kidney (OK) cells, a model of renal proximal tubule. PTH 1–34 100 nM increased phosphorylation of cPLA2 in OK cells as determined by immunoblot with phosphoPLA2 antibodies. PTH-stimulated phosphorylation of cPLA2 was prevented by pretreatment with U0126, an inhibitor of MEK1, but not by a peptide inhibitor of PKC alpha. Treatment of OK cells with 20-HETE or arachidonic acid (AA) (products of cPLA2) decreased Na-K activity by 25–30% as measured by 86Rb uptake and decreased Na-K membrane expression as demonstrated by decreased biotinylated Na-K. The effects of 20-HETE and AA were blocked by the peptide inhibitor of PKC alpha but not by U0126. Immunoprecipitation of cell lysates with Na-K alpha subunit antibody followed by immunoblot for PKC alpha confirmed that 20-HETE and AA increased PKC alpha association with Na-K. We conclude that PTH regulates Na-K in OK cells through an ERK-dependent cPLA2 signaling pathway.

Erythrocyte sodium pump stimulation by ouabain and an endogenous ouabain‐like factor

Cardiac glycosides inhibit the sodium pump. However, some studies suggest that nanomolar ouabain concentrations can stimulate the activity of the sodium pump. In this study, using the Na(+)/K(+)-ATPase of human erythrocytes, we compared the effect of digoxin, ouabain and an ouabain like-factor (OLF), on (86)Rb uptake. Ouabain concentrations below 10(-9) M significantly stimulate Rb(+) uptake, and the maximal increase above base-line values is 18 +/- 5% at 10(-10) M ouabain. No stimulation is observed in the same conditions by digoxin. OLF behaved like ouabain, producing an activation of Rb(+) flux at concentrations lower than 10(-9) M ouabain equivalents (14 +/- 3% at 10(-10) M). Western blot analysis revealed the presence of both alpha(1) and alpha(3) pump isoforms in human erythrocytes. Our data confirm the analogies between OLF and ouabain and suggest that Na(+)/K(+)-ATPase activation may be related to the alpha(3) isoform. In addition, we investigated whether ouabain at different concentrations was effective in altering the intracellular calcium concentration of erythrocytes. We found that ouabain at concentration lower than 10(-9) M did not affect this homeostasis.

Functional Comparison of Red Cell KCl Cotransporter Isoforms, KCC1, KCC3, and KCC4.

The KCl cotransporter mediates volume reduction in normal (AA) reticulocytes, and its abnormal regulation in sickle (SS) reticulocytes contributes to cellular dehydration that facilitates Hb S sickling. mRNA for three KCC genes - KCC1, KCC3, and KCC4 - as well as a splicing isoform, KCC1ex1b, is present in reticulocytes (Exp. Hem. 2005; 33:624). Western blotting has demonstrated KCC1 in human RBC membranes (Su et al, AJPhysiol. 1999; 277:C899) and KCC3 in sheep (Lauf et al, CompBiochemPhysiol 2001; 130:499); KCC4 protein was found in hRBC membranes by proteomic analysis (Pasini et al, Blood 2006; 108:791). We confirm here the presence of KCC3 protein in hRBC via western blotting using an antibody to an exon 3 epitope distal to known N-terminal splicing sites. We sought to characterize and compare human KCC isoforms expressed in human cells (HEK 293) and assess their similarity to KCC activity in RBC. cDNAs for human KCC1, KCC1ex1b, KCC3a, and KCC4, with N-terminal c-myc epitope tags were expressed in HEK 293 cells. Stable cell lines were selected by growth in neomycin, and expression monitored by quantitative PCR analysis of the expressed construct and other endogenous isoforms and by western blotting (anti-myc) of plasma membranes. KCC activity was measured as N-ethylmaleimide(NEM)-stimulated, Cl-dependent Rb uptake in cells grown to 75–90 % confluency. Cells were incubated at 37°C in isotonic saline media with various concentrations of Rb (Na replacement), plus 0.1 mM ouabain and 0.01 mM bumetanide. At 2 and 4 min cells were washed with iced Rb-free media, then lysed and assayed for Rb by flame emission, normalized to sample protein. Flux rates were calculated from Rb uptake at 2 and 4 min. The flux rate in Cl-free sulfamate media was subtracted from that in Cl-media to yield the Cl-dependent flux rate. Wild-type HEK cells showed no increase in Cl-dependent Rb influx when exposed to 1 mM NEM, but Cl-dependent, NEM-stimulated Rb uptake was apparent in cells expressing KCC isoforms. All isoforms were stimulated by hypotonic conditions (75 mOsm), but relative to NEM-stimulated activity, KCC3 was most responsive. Kinetic characteristics of Cl-dependent, NEM-stimulated Rb influx in HEK cell expressing human KCC1, KCC3a, and KCC4 isoforms isoforms are given below, compared to RBC KCC fluxes. A splice variant, KCC1ex1b, coding for a protein with a truncated N-terminus, exhibited 48 ± 7% of the activity of the full-length KCC1 isoform, but did not alter transport activity of coexpressed KCC3a. Thus, KCC isoforms expressed in HEK cells share some, but not all, characteristics with RBC KCC fluxes. The isoform kinetics also differ from those previously published in xenopus oocyte expression systems, suggesting that the membrane milieu in which KCC proteins are expressed may influence their functional characteristics. Likewise, KCC activity in RBC may represent an average of several transporters operating in parallel. The truncated KCC1ex1b isoform, expressed at higher levels in normal than in sickle reticulocytes, has lower activity than KCC1. KCC3 and KCC4 exhibit more robust transport activity than KCC1 and may mediate a substantial part of KCC activity in RBC.KCC Kinetic CharacteristicsVMax (μmol/mgprot./min)Km (ext. Rb, mM)Anion SelectivityhKCC128.9 ± 5.616.7 ± 5.4Cl > Br > I > SCNhKCC3a107 ± 4511.0 ± 7.1Cl = Br > I > SCNhKCC4206 ± 1615.6 ± 8.2Cl > Br >> I = SCNRBCNA12 - 20Cl = Br >> I = SCN