Plasma-membrane electrical responses to salt and osmotic gradients contradict radiotracer kinetics, and reveal Na+-transport dynamics in rice (Oryza sativa L.).

MAIN CONCLUSION: A systematic analysis of NaCl-dependent, plasma-membrane depolarization (∆∆Ψ) in rice roots calls into question the current leading model of rapid membrane cycling of Na+ under salt stress. To investigate the character and mechanisms of Na+ influx into roots, Na+-dependent changes in plasma-membrane electrical potentials (∆∆Ψ) were measured in root cells of intact rice (Oryza sativa L., cv. Pokkali) seedlings. As external sodium concentrations ([Na+]ext) were increased in a step gradient from 0 to 100 mM, membrane potentials depolarized in a saturable manner, fitting a Michaelis-Menten model and contradicting the linear (non-saturating) models developed from radiotracer studies. Clear differences in saturation patterns were found between plants grown under low- and high-nutrient (LN and HN) conditions, with LN plants showing greater depolarization and higher affinity for Na+ (i.e., higher Vmax and lower Km) than HN plants. In addition, counterion effects on ∆∆Ψ were pronounced in LN plants (with ∆∆Ψ decreasing in the order: Cl- > SO42- > HPO 4 2- ), but not seen in HN plants. When effects of osmotic strength, Cl- influx, K+ efflux, and H+-ATPase activity on ∆∆Ψ were accounted for, resultant Km and Vmax values suggested that a single, dominant Na+-transport mechanism was operating under each nutritional condition, with Km values of 1.2 and 16 mM for LN and HN plants, respectively. Comparing saturating patterns of depolarization to linear patterns of 24Na+ radiotracer influx leads to the conclusion that electrophysiological and tracer methods do not report the same phenomena and that the current model of rapid transmembrane sodium cycling may require revision.

Evidence that Na+/H+ exchanger 1 is an ATP-binding protein.

Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1.

The death of ouabain-treated renal epithelial C11-MDCK cells is not mediated by swelling-induced plasma membrane rupture.

This study examined the role of cell volume modulation in plasma membrane rupture and death documented in ouabain-treated renal epithelial cells. Long-term exposure to ouabain caused massive death of C11-MDCK (Madin-Darby canine kidney) epithelial cells, documented by their detachment, chromatin cleavage and complete loss of lactate dehydrogenase (LDH), but did not affect the survival of vascular smooth muscle cells (VSMCs) from the rat aorta. Unlike the distinct impact on cell survival, 2-h exposure to ouabain led to sharp elevation of the [Na⁺](i)/[K⁺](i) ratio in both cell types. A similar increment of Na⁺(i) content was evoked by sustained inhibition of Na⁺,K⁺-ATPase in K⁺-free medium. However, in contrast to ouabain, C11-MDCK cells survived perfectly during 24-h exposure to K⁺-free medium. At 3 h, the volume of ouabain-treated C11-MDCK cells and VSMCs, measured by the recently developed dual-image surface reconstruction technique, was increased by 16 and 12%, respectively, whereas 5-10 min before the detachment of ouabain-treated C11-MDCK cells, their volume was augmented by ~30-40%. To examine the role of modest swelling in the plasma membrane rupture of ouabain-treated cells, we compared actions of hypotonic medium on volume and LDH release. We observed that LDH release from hypoosmotically swollen C11-MDCK cells was triggered when their volume was increased by approximately fivefold. Thus, our results showed that the rupture of plasma membranes in ouabain-treated C11-MDCK cells was not directly caused by cell volume modulation evoked by Na⁺,K⁺-ATPase inhibition and inversion of the [Na⁺](i)/[K⁺](i) ratio.

BmKCT toxin inhibits glioma proliferation and tumor metastasis.

Malignant gliomas are the most common primary brain tumors associated with significant morbidity and mortality. How to target the tumor in situ, and inhibit tumor cell proliferation and invasion is the key for therapy. Gliomas express a glioma-specific chloride ion channel that is sensitive to toxins including BmKCT. In the current study, the inhibitory effect of BmKCT on glioma growth was observed in vivo using the glioma/SD rat model. Furthermore, BmKCT prevented the metastasis of glioma cells in vivo. Moreover, biodistribution experiments with (l3l)I-labeled or Cy5.5-conjugated BmKCT revealed that BmKCT selectively targeted the glioma in situ. Our data suggest that BmKCT could be exploited as a potential therapeutic for glioma diagnosis and therapy.

Characterization of transport mechanisms and determinants critical for Na+-dependent Pi symport of the PiT family paralogs human PiT1 and PiT2.

The general phosphate need in mammalian cells is accommodated by members of the P(i) transport (PiT) family (SLC20), which use either Na(+) or H(+) to mediate inorganic phosphate (P(i)) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na(+)-dependent P(i) (NaP(i)) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with (32)P(i) as a traceable P(i) source. For PiT1, the Michaelis-Menten constant for P(i) was determined as 322.5 +/- 124.5 microM. PiT2 was analyzed for the first time and showed positive cooperativity in P(i) uptake with a half-maximal activity constant for P(i) of 163.5 +/- 39.8 microM. PiT1- and PiT2-mediated Na(+)-dependent P(i) uptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na(+) dependency patterns. However, only PiT2 was capable of Na(+)-independent P(i) transport at acidic pH. Study of the impact of divalent cations Ca(2+) and Mg(2+) revealed that Ca(2+) was important, but not critical, for NaP(i) transport function of PiT proteins. To gain insight into the NaP(i) cotransport function, we analyzed PiT2 and a PiT2 P(i) transport knockout mutant using (22)Na(+) as a traceable Na(+) source. Na(+) was transported by PiT2 even without P(i) in the uptake medium and also when P(i) transport function was knocked out. This is the first time decoupling of P(i) from Na(+) transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E(55) and E(575) are responsible for linking P(i) import to Na(+) transport in PiT2.

Effect of sensitization on membrane ion fluxes & intracellular calcium in guineapigs.

BACKGROUND & OBJECTIVES: The biochemical mechanisms underlying the development of sensitization-induced airway hyperresponsiveness (AHR) in asthma are poorly defined. Alterations in the regulation of intracellular calcium may play an important role in its pathogenesis. We carried out this study to see the effect of sensitization with ovalbumin on membrane ion fluxes and intracellular calcium in a guinea pig model. METHODS: Airway reactivity to inhaled histamine was measured initially and after sensitization with ovalbumin in 28 guineapigs. Intracellular calcium [Ca(2+)]i was measured in tracheal smooth muscle cells and peripheral leukocytes using fluorescent dye FURA 2AM. Calcium and sodium ion influx across the cell membrane was measured in leukocytes. Ouabain-sensitive Rubidium ((86)Rb) influx was measured in tracheal smooth muscles cells. The activities of Na(+), K(+) ATPase and Ca(2+) ATPase were measured in tracheal smooth muscle cells. Lipid peroxides were measured in plasma. RESULTS: Airway responsiveness was significantly (P<0.001) increased after sensitization along with an increase in [Ca2+]i levels in leukocytes and tracheal smooth muscle cells, higher rates of (45)Ca and (22)Na influx in leukocytes and higher (86)Rb influx rates in tracheal smooth muscle cells, and increased levels of lipid peroxides in plasma. INTERPRETATION & CONCLUSION: In guineapig model of asthma sensitization to allergen increased the membrane permeability to calcium and sodium, and intracellular calcium levels. These alterations may play a role in the pathogenesis of airway hyper-responsiveness following sensitization.

Replacement of (alpha)1-Na-K-ATPase of Dahl rats by Milan rats lowers blood pressure but does not affect its activity.

Both linkage and use of congenic strains have shown that a chromosome region near the gene for the Na-K-ATPase alpha(1)-subunit (Atp1a1) contained a quantitative trait locus (QTL) for blood pressure (BP). Currently, two congenic strains, designated S.M5 and S.M6, were made by replacing a segment of the Dahl salt-sensitive SS/Jr (S) rat by the homologous region of the Milan normotensive rat (MNS). In S.M5, the gene for Atp1a1 is from the MNS strain; whereas in S.M6, Atp1a1 is from the S strain. The baseline activity of the alpha(1)-Na-K-ATPase and its stoichiometry were evaluated by an assay of ouabain-sensitive inwardly and outwardly directed (86)Rb and (22)Na fluxes in erythrocytes. The two congenic strains showed a similar BP, but both had a BP lower than that of S rats (P < 0.0001). Neither the alpha(1)-Na-K-ATPase activity nor its stoichiometry was affected by the substitution of the Atp1a1 alleles of S by those of MNS. Thus the BP-lowering effects observed in S.M5 and S.M6 could not be attributed to the alpha(1)-Na-K-ATPase activity or its stoichiometry. Atp1a1 is not supported as a candidate to be a BP QTL.

Morphological and functional changes in the colonic epithelial cells in a rabbit model of colitis.

A rabbit model of TNBS-colitis was used to study the effect of intestinal inflammation on epithelial cell function. Epithelial cells were isolated using a non-enzymatic isolation method without any apparent contamination with infiltrating immune cells. The isolated cells were found to be viable using dye exclusion studies, unidirectional Na+ -fluxes, proliferation assays and morphological studies. The cells, however, showed morphological changes that suggested the presence of increased number of secretory vesicles. This increase correlated well with the increase observed in ion and water secretion as measured by the short-circuit current. Finally, in the colitic tissue the number of PGE2 receptors was greatly reduced with no changes observed in the affinity of PGE2 to its receptor. The reduced number of PGE2 receptors might be due to sensitization of the receptor. In conclusion, we have demonstrated that morphologically and functionally normal epithelial cells can be isolated from the rabbit inflamed distal colon.

Voltage-generated torque drives the motor of the ATP synthase.

The mechanism by which ion-flux through the membrane-bound motor module (F0) induces rotational torque, driving the rotation of the gamma subunit, was probed with a Na+-translocating hybrid ATP synthase. The ATP-dependent occlusion of 1 (22)Na+ per ATP synthase persisted after modification of the c subunit ring with dicyclohexylcarbodiimide (DCCD), when 22Na+ was added first and ATP second, but not if the order of addition was reversed. These results support the model of ATP-driven rotation of the c subunit oligomer (rotor) versus subunit a (stator) that stops when either a 22Na+-loaded or a DCCD-modified rotor subunit reaches the Na+-impermeable stator. The ATP synthase with a Na+-permeable stator catalyzed 22Na+out/Na+in-exchange after reconstitution into proteoliposomes, which was not significantly affected by DCCD modification of the c subunit oligomer, but was abolished by the additional presence of ATP or by a membrane potential (DeltaPsi) of 90 mV. We propose that in the idling mode of the motor, Na+ ions are shuttled across the membrane by limited back and forth movements of the rotor against the stator. This motional flexibility is arrested if either ATP or DeltaPsi induces the switch from idling into a directed rotation. The Propionigenium modestum ATP synthase catalyzed ATP formation with DeltaPsi of 60-125 mV but not with DeltapNa+ of 195 mV. These results demonstrate that electric forces are essential for ATP synthesis and lead to a new concept of rotary-torque generation in the ATP synthase motor.

Differential regulation of Na+ and Cl- conductances by PTX-sensitive G proteins in fetal lung apical membrane vesicles.

In apical membrane vesicles (AMV) prepared from late gestation fetal guinea pig lung we show that conductive 22Na+ uptake is modulated by at least two pathways involving pertussis toxin (PTX)-sensitive G proteins. Intravesicular incorporation of 100 microM GTPgammaS into vesicles resuspended in NaCl caused a significant stimulation (P<0. 05) of conductive Na+ uptake in AMV to 150+/-10% (n=10) of control, whereas GDPbetaS reduced uptake to 65+/-9% (n=4) of control. This contrasting response to GTPgammaS and GDPbetaS is characteristic of a G protein mediated pathway. GTPgammaS induced a significantly smaller stimulation, 125+/-8% (n=5) of control, in the presence of the relatively impermeant anion isethionate (Ise-). Taken together, these data indicate modulation of both Na+ and Cl- channels in the apical membrane by co-localised G protein(s). Treatment with PTX stimulated conductive 22Na+ uptake to 171+/-20% (n=13) of control in AMV resuspended in NaCl, but did not have a significant effect, 94+/-19% of control, in the presence of NaIse indicating the existence of tonic activation of Cl- channels in these AMV under resting conditions. As the combined effects of PTX and GTPgammaS diminished uptake, we propose that the G protein(s) responsible for Na+ channel activation in response to GTPgammaS is PTX-sensitive and that additional PTX-insensitive G proteins might also modulate 22Na+ uptake in these AMV. The presence of Gialpha1, Gialpha2, Gialpha3 and Goalpha in this apical membrane preparation was confirmed by PTX catalysed [32P]ADP-dependent ribosylation and Western blotting. Incubation of AMV with 200 microM DTT caused an inhibition of conductive Na+ uptake in AMV resuspended in NaCl or NaIse to 66+/-8% (n=11) and 64+/-8% (n=6) of control respectively. Pre-treatment with DTT did not affect the ability of GTPgammaS to stimulate conductive Na+ uptake suggesting that the regulation of 22Na+ uptake in late gestation guinea pig fetal lung AMV is unlikely to involve an associated regulatory protein.

Effects of high glucose on the production of heparan sulfate proteoglycan by mesangial and epithelial cells.

Changes in heparan sulfate metabolism may be important in the pathogenesis of diabetic nephropathy. Recent studies performed on renal biopsies from patients with diabetic nephropathy revealed a decrease in heparan sulfate glycosaminoglycan staining in the glomerular basement membrane without changes in staining for heparan sulfate proteoglycan-core protein. To understand this phenomenon at the cellular level, we investigated the effect of high glucose conditions on the synthesis of heparan sulfate proteoglycan by glomerular cells in vitro. Human adult mesangial and glomerular visceral epithelial cells were cultured under normal (5 mM) and high glucose (25 mM) conditions. Immunofluorescence performed on cells cultured in 25 mM glucose confirmed and extended the in vivo histological observations. Using metabolic labeling we observed an altered proteoglycan production under high glucose conditions, with predominantly a decrease in heparan sulfate compared to dermatan sulfate or chondroitin sulfate proteoglycan. N-sulfation analysis of heparan sulfate proteoglycan produced under high glucose conditions revealed less di- and tetrasaccharides compared to larger oligosaccharides, indicating an altered sulfation pattern. Furthermore, with quantification of glomerular basement membrane heparan sulfate by ELISA, a significant decrease was observed when mesangial and visceral epithelial cells were cultured in high glucose conditions. We conclude that high glucose concentration induces a significant alteration of heparan sulfate production by mesangial cells and visceral epithelial cells. Changes in sulfation and changes in absolute quantities are both observed and may explain the earlier in vivo observations. These changes may be of importance for the altered integrity of the glomerular charge-dependent filtration barrier and growth-factor matrix interactions in diabetic nephropathy.

Stimulation of granulocytic cell iodination by pine cone antitumor substances.

Antitumor substances (Fractions VI and VII) prepared from the NaOH extract of pine cone significantly stimulated the iodination (incorporation of radioactive iodine into an acid-insoluble fraction) of human peripheral blood adherent mononuclear cells, polymorphonuclear cells (PMN), and human promyelocytic leukemic HL-60 cells. In contrast, these fractions did not significantly increase the iodination of nonadherent mononuclear cells, red blood cells, other human leukemic cell lines (U-937, THP-1, K-562), human diploid fibroblast (UT20Lu), or mouse cell lines (L-929, J774.1). Iodination of HL-60 cells, which were induced to differentiate by treatment with either retinoic acid or tumor necrosis factor, were stimulated less than untreated cells. The stimulation of iodination of both PMN and HL-60 cells required the continuous presence of these fractions and was almost completely abolished by the presence of myeloperoxidase inhibitors. The stimulation activity of these fractions was generally higher than that of various other immunopotentiators. Possible mechanisms of extract stimulation of myeloperoxidase-containing cell iodination are discussed.

Biodistributions of radioactive alkaline metals in tumor bearing animals: comparison with 201Tl.

The retention values for 42K, 86Rb and 134Cs in the tissues and blood were quite similar to those for 201Tl, but were very different from those for 22Na. In an experiment for subcellular fractionation of tumors, most of these nuclides were localized in the supernatant fraction, with small amounts in other fractions. The concentration ratios for these nuclides in each fraction were approximately constant regardless of the time after administration. Radioactive alkaline metals in the supernatant fraction of the tumor homogenate existed mostly as free ions and were bound to protein in other fractions of tumor tissue. These results were essentially the same as those for 201Tl. Ouabain suppression studies indicated that 201Tl is taken up into the tumor cells partly through Na+, K+-ATPase of their membranes. Ionic radii of alkaline metals and thallium were related to their blood and tumor retention values. This relationship suggested that monovalent cations whose ionic radii exceed 0.133 nm, and which exist as free ions in the tissue fluids, behave like the potassium ion. Potassium and K analogs (Tl, Rb, Cs) are avidly taken up into viable tumor cells whose Na+, K+-ATPase activity is elevated. Therefore, suitable radionuclides of K and K analogs can be excellent agents for visualization of viable tumor tissues.

Effects of chlorpromazine on Na+-K+-ATPase pumping and solute transport in rat hepatocytes.

Inhibition of Na+-K+-ATPase and sodium-dependent bile acid transport has been suggested as a mechanism for the cholestasis produced by certain drugs such as chlorpromazine. We examined the effects of chlorpromazine (and in selected studies, two of its metabolites) on Na+-K+-ATPase cation pumping (ouabain-suppressible 86Rb uptake), exchangeable intracellular sodium content, membrane potential (assessed by 36Cl- distribution), and sodium-dependent transport of taurocholate and alanine in primary cultures of rat hepatocytes. Chlorpromazine (10-300 microM), 7,8-dihydroxychlorpromazine (10-300 microM), and ouabain (0.1-2 mM), but not chlorpromazine sulfoxide, produced a concentration-dependent decrease in Na+-K+-ATPase cation pumping and an increase in intracellular sodium content. Chlorpromazine (100 microM) and ouabain (0.75 mM) also modestly decreased hepatocyte membrane potential. In further studies, chlorpromazine (75 and 100 microM) and ouabain (0.1, 0.5, and 0.75 mM) decreased initial sodium-dependent uptake rates of taurocholate and alanine by 18-63%. Although the steady-state intracellular content of alanine was decreased 25-53% by both agents, chlorpromazine increased the steady-state content of taurocholate by 171% and decreased taurocholate efflux, apparently related to partitioning of taurocholate into a large, slowly turning over intracellular pool. These studies provide direct evidence that chlorpromazine inhibits Na+-K+-ATPase cation pumping in intact cells and that partial inhibition of Na+-K+-ATPase cation pumping is associated with a reduction of both the electrochemical sodium gradient and sodium-dependent solute transport. These effects of chlorpromazine may contribute to chlorpromazine-induced cholestasis in animals and humans.

The effect of ouabain on placental transport of 86Rb.

Human placental fragments concentrate 86Rb 10--20-fold during a two-hour incubation period. Inhibition of ouabain is dose-dependent, reaching 90 + per cent at a concentration of 5 x 10(-5) M. The clearance index of 86Rb across the perfused human placenta is 0.34 +/- 0.08, comparing to previously reported indices for Na22 and Cl36 of 0.28 and 0.41, respectively. Ouabain in concentrations up to 5 x 10(-5) M had no detectable effect on transfer across the placenta. The clearance index of ouabain is low, averaging 0.07 in 3 experiments. 3H-ouabain is not detectably bound to albumin or placental homogenate.

Influence of glucose on Ehrlich cell volume, ion transport, and membrane potential.

Incubating Ehrlich ascites tumor cells with 10 mM glucose at room temperature resulted in the following changes. The cells shrank, reaching a minimum volume after 1 h. The decrease in cell volume was 50-90% inhibited by 1 mM furosemide. The mmol K+ and Cl-/mg dry wt decreased, and mmol Na+/mg dry wt increased over the 1 h incubation. The net loss of KCl was inhibited by 1 mM furosemide. Immediately after the addition of glucose, the influx of 86Rb sensitive to ouabain decreased, whereas the influx sensitive to furosemide increased. The total influx of 86Rb with glucose was similar to that of controls. The effluxes of 86Rb and 36Cl increased immediately after the addition of glucose. These effluxes did not increase, however, in the presence of 1 mM furosemide. Initially the ouabain-sensitive Na+ efflux was not changed with glucose, but the ouabain-insensitive Na+ efflux decreased. Furosemide (1 mM) did not influence Na+ efflux. With time the ouabain-sensitive Na+ efflux increased as cellular Na+ levels rose so that at 1 h the ouabain-sensitive Na+ efflux from glucose-treated cells was 2.5-3 times that of control cells. The potential difference across the membrane gradually became more negative by approximately 25 mV, reaching a maximum after 1 h. The hyperpolarization was reversed by 1 mM ouabain. The changes in ionic fluxes on the addition of glucose are compared with changes in ionic fluxes seen during volume regulation.