Overexpression of Na+/K+-ATPase Parallels the Increase in Sodium Transport and Potassium Recycling in an In Vitro Model of Proximal Tubule Cellular Ageing

Na⁺/K⁺-ATPase plays a key role in the transport of Na⁺ throughout the nephron, but ageing appears to be accompanied by changes in the regulation and localization of the pump. In the present study, we examined the effect of in vitro cell ageing on the transport of Na⁺ and K⁺ ions in opossum kidney (OK) cells in culture. Cells were aged by repeated passing, and Na⁺/K⁺-ATPase activity and K⁺ conductance were evaluated using electrophysiological methods. Na⁺K⁺-ATPase α₁– and β₁-subunit expression was quantified by Western blot techniques. Na⁺/H⁺ exchanger activity, changes in membrane potential, cell viability, hydrogen peroxide production and cellular proliferation were determined using fluorimetric assays. In vitro cell ageing is accompanied by an increase in transepithelial Na⁺ transport, which results from an increase in the number of Na⁺/K⁺-ATPase α₁- and β₁-subunits, in the membrane. Increases in Na⁺/K⁺-ATPase activity were accompanied by increases in K⁺ conductance as a result of functional coupling between Na⁺/K⁺-ATPase and basolateral K⁺ channels. Cell depolarization induced by both KCl and ouabain was more pronounced in aged cells. No changes in Na⁺/H⁺ exchanger activity were observed. H₂O₂ production was increased in aged cells, but exposure for 5 days to 1 and 10 μM of H₂O₂ had no effect on Na⁺/K⁺-ATPase expression. Ouabain (100 nM) increased α₁-subunit, but not β₁-subunit, Na⁺/K⁺-ATPase expression in aged cells only. These cells constitute an interesting model for the study of renal epithelial cell ageing.     

Effect of sodium on cellular calcium transport in rat kidney

Summary The influence of extracellular Na (Na _o ) on cellular Ca transport and distribution was studied in rat kidney slices. Calcium efflux from prelabeled slices was depressed when Na _o was completely replaced by choline or tetraethylammonium (TEA) ions and it was markedly stimulated when Na was reintroduced in a Na-free medium. However, reducing Na _o (with choline or TEA as substituting ions) did not increase the total slice⁴⁰Ca, their total exchangeable Ca pool, or the⁴⁰Ca or⁴⁵Ca of mitochondria isolated from these slices. Kinetic analyses of steady-state⁴⁵Ca desaturation curves showed that reducing Na _o depressed the exchange of Ca across the plasma membrane, slightly decreased the cytosolic Ca pool, but did not significantly affect the mitochondrial Ca pool and Ca cycling. Ouabain (10^–3 m) which should reduce the Na gradient across the plasma membrane had no effect on calcium distribution and transport. These results suggest that in kidney cells low Na _o depresses Ca influx as well as Ca efflux; there may be an interaction between Na and Ca at a possible carrier located in the plasma membrane, but there is no Na/Ca exchange as described in several excitable tissues.     

Calcium channels play a pivotal role in the sequence of ionic changes involved in initiation of mouse sperm acrosomal exocytosis

The sequence of ionic changes involved in initiation of acrosomal exocytosis in capacitated mouse spermatozoa was investigated. Earlier studies demonstrated that a large influx of Na⁺ is required for exocytosis, this Na⁺ apparently being associated with an increase in intracellular pH (pH_i) via an Na⁺-H⁺ exchanger. This rise in pH_i may in turn activate calcium channels and permit the influx of extracellular Ca²⁺ needed to trigger acrosomal exocytosis. In the present study, the dihydropyridine voltage-dependent calcium channel antagonist nifedipine was able to inhibit significantly exocytosis in sperm cells treated in various ways capable of stimulating acrosomal loss. The monovalent cation ionophore monensin can promote Na⁺ entry required for both capacitation and acrosomal exocytosis, as demonstrated by using chlortetracycline to monitor changes in sperm functional potential. In the presence of 10 nM nifedipine, monensin treatment accelerated capacitation but was unable to trigger exocytosis. The requirement for internalization of a high concentration of Na⁺ can be bypassed by the addition of 25 mM NH₄CI to raise the pH_i of cells capacitated in 25NH₄CI to raise the pH_i of cells capacitated in 25 mM Na⁺ (insufficient Na⁺ to support exocytosis under usual conditions). Again, introduction of nifedipine was able to inhibit exocytosis. In a third experimental approach, amiloridestimulated exocytosis in capacitated cells was significantly inhibited by nifedipine. In contrast to these treatments directed at specific mechanisms, the ability of the Ca²⁺ inophore A23187 to promote more general entry of Ca²⁺ and thereby to accelerate capacitation and exocytosis was not inhibited by nifedipine. Finally, monensin-treated cells exhibited a rise and then a fall in ⁴⁵Ca²⁺ uptake, the time course of which paralleled stimulation of acrosomal exocytosis in similarly treated cells. Nifedipine significantly reduced this uptake. The fact that nifedipine can block exocytosis induced by a variety treatments strongly suggests that voltage-dependent calcium channels play a pivotal role in the response. These results are consistent with the following sequence of ionic changes in capacitated cells leading to acrosomal exocytosis: [Na⁺]_i ↑ → [H]_i↓ → pH_i ↑ → activation of calcium channels → [Ca²⁺]_i ↑ → exocytosis. Given that zona-induced exocytosis is reportedly an indirect response, mediated by voltage-dependent calcium channels, and that the Na⁺-H⁺ exchanger in somatic cells can be activated by receptor-mediated mechanisms, we suggest that sperm-zona inter action promotes an influx of Na⁺ by activating an Na⁺-H⁺ exchanger and thereby initiating the above sequence of changes. © 1993 Wiley-Liss, Inc.     

Structure-activity relationship of amiloride analogs as blockers of epithelial Na channels: II. Side-chain modifications

Summary The overall on- and off-rate constants for blockage of epithelial Na channels by amiloride analogs were estimated by noise analysis of the stationary Na current traversing frog skin epithelium. The (2-position) side chain structure of amiloride was varied in order to obtain structure/rate constant relationships. (1) Hydrophobic chain elongations (benzamil and related compounds of high blocking potency) increase the stability of the blocking complex (lowered off-rate), explained by attachment of the added phenyl moiety to a hydrophobic area near the site of side chain interaction with the channel protein. (2) Some other chain modifications show that the on-rate, which is smaller than a diffusion-limited rate, varies with side chain structure. In several cases this effect is not attributable to steric hindrance on encounter, and implies that the side chain interacts briefly with the channel protein (encounter complex) before the main blocking position of the molecule is attained. The encounter complex must be labile since the overall rate constants of blockage are not concentration-dependent. (3) In two cases, changes at the 2-position side chain and at other ring ligands, with known effects on the blocking rate constants, could be combined in one analog. The rate constants of blocking by the resulting compounds indicate that the structural changes have additive effects in terms of activation energies. (4) Along with other observations (voltage dependence of the rate constants and competition with the transported Na ion), these results suggest a blocking process of at least two steps. It appears that initially the 2-position side chain invades the outward-facing channel entrance, establishing a labile complex. Then the molecule is either released completely (no block) or the 6-ligand of the pyrazine ring gains access to its receptor counterpart, thus establishing the blocking complex, the lifetime of which is strongly determined by the electronegativity of the 6-ligand.     

Optical study of active ion transport in lipid vesicles containing reconstituted Na,K-ATPase

Summary A fluorescence method is described for the measurement of ATP-driven ion fluxes in lipid vesicles containing purified Na,K-ATPase. The membrane voltage of enzyme containing vesicles was measured by using a voltage-sensitive indocyanine dye. By addition of valinomycin the vesicle membrane is made selectively permeable to K⁺ so that the membrane voltage approaches the Nernst potential for K⁺. With constant external K⁺ concentration, the time course of internal K⁺ concentration can be continuously measured as change of the fluorescence signal after activation of the pump. The optical method has a higher time resolution than tracer-flux experiments and allows an accurate determination of initial flux rates. From the temperature dependence of active K⁺ transport its activation energy was determined to be 115 kJ/mol. ATP-stimulated electrogenic pumping can be measured as a fast fluorescence change when the membrane conductance is low (i.e., at low or zero valinomycin concentration). In accordance with expectation, the amplitude of the fast signal change increases with decreasing passive ion permeability of the vesicle membrane. The resolution of the charge movement is so high that a few pump turnovers can be easily detected.     

Sodium flux in the apical membrane of the toad skin: Aspects of its regulation and the importance of the ionic strength of the outer solution upon the reversibility of amiloride inhibition

Summary Injection of small pulses of concentrate solutions of salts or drugs into the outer bathing fluid led to sudden increases of its solute concentration. Vigorous stirring of the outer bathing solution was used to minimize the thickness of the unstirred layer adjacent to the outer skin surface. Pulses of 1m NaCl injected into the outer compartment induced sharp increases of the SCC following a time course variable with the magnitude of the pulse and the particular condition of each skin. Comparison of the spontaneous decline of the SCC with the decline induced by a small dose of amiloride, where an increase inR was observed, indicates that the spontaneous decline cannot be explained simply as a reduction of the Na permeability of the apical membrane by self-inhibition of feedback inhibition of the apical membrane Na channels. Reduction of the driving force for Na movement into the epithelial cells must play an important role in the process. Reversibility of the amiloride inhibition of the SCC was highly dependent upon the ionic strength of the solution used to rinse and wash out the inhibitor from the outer skin surface. With H₂O, the amiloride molecules washed out slowly as compared to NaCl or KCl solutions. Na or K have the same ability to dislodge the amiloride molecules from their binding sites. This effect is apparently of a purely electrostatic nature.     

Intracellular ion concentrations in the isolated frog skin epithelium: Evidence for different types of mitochondria-rich cells

Summary Intracellular ion concentrations were determined in split skins of Rana pipiens using the technique of electron microprobe analysis. Under control conditions, principal cells and mitochondria-rich cells (MR cells) had a similar intracellular ion composition, only the Cl concentration in MR cells was significantly lower. Inhibition of transepithelial Na transport by low concentrations of ouabain (2 × 10^–6 m, innerbath) resulted in a Na concentration increase of principal cells from 10.9 to 54.3 mmol/kg wet wt. The increase was completely abolished by simultaneous application of amiloride (10^–4 m, outer bath). Amiloride alone resulted in a significant decrease of the Na concentration to 6.1 mmol/kg. w. w. Among MR cells, two different groups of cells could be distinguished; cells that showed a Na increase after ouabain which was even larger than that in principal cells and cells that did not respond to ouabain. In about half of all ouabain-sensitive MR cells the Na increase could be prevented by amiloride. According to these results, a subpopulation of MR cells displays the transport characteristics expected for a transepithelial Na transport compartment, an apical amiloride-sensitive Na influx and abasal ouabain-inhibitable Na efflux. Given the small number of cells, however, it is unlikely that this subtype of MR cells contributes significantly to the overall rate of transepithelial Na transport.I wish to thank Cathy Langford, Cindy Partain, and Ray Whitfield for their excellent technical assistance. Financial support was provided by NIH grants DK35717 and 1S10-RR0-234501.     

Interactions of amiloride and other blocking cations with the apical Na channel in the toad urinary bladder

Summary A simple model of the action of amiloride to block apical Na channels in the toad urinary bladder was tested. According to the model, the positively charged form of the drug binds to a site in the lumen of the channel within the electric field of the membrane. In agreement with the predictions of the model: (1) The voltage dependence of amiloride block was consistent with the assumption of a single amiloride binding site, at which about 15% of the transmembrane voltage is sensed, over a voltage range of ±160 mV. (2) The time course of the development of voltage dependence was consistent with that predicted from the rate constants for amiloride binding previously determined. (3) The ability of organic cations to mimic the action of amiloride showed a size dependence implying a restriction of access to the binding site, with an effective diameter of about 5 angstroms. In a fourth test, divalent cations (Ca, Mg, Ba and Sr) were found to block Na channels with a complex voltage dependence, suggesting that these ions interact with two or more sites. at least one of which may be within the lumen of the pore.     

Potassium/proton exchange in brush-border membrane of rat ileum

Summary These experiments were designed to determine whether proton-driven⁸⁶Rb uptake was present in apical membrane vesicles prepared from rat ileum. The uptake of⁸⁶Rb was approximately 300 to 350% greater in the presence of a 100-fold H⁺ gradient than in its absence and was greater at 1, 2 and 5 minutes (overshoot) than that at 90 minutes. Proton-driven⁸⁶Rb uptake was decreased by 20% in TMA-nitrate compared to that in TMA-gluconate. 0.3mm amiloride did not significantly inhibit proton-driven⁸⁶Rb uptake; in contrast, proton-driven²²Na uptake was significantly inhibited by 0.3mm amiloride by 34%. Similarly, 25mm KCl inhibited proton-driven⁸⁶Rb uptake more than that of²²Na, while the inhibition of proton-driven²²Na uptake by 25mm NaCl was greater than that of⁸⁶Rb. In additional studies intravesicular acidification measured by acridine orange fluorescence was demonstrated in the presence of an out-wardly directed K gradient. These studies demonstrate that a proton gradient stimulates⁸⁶Rb uptake and a K gradient induces intravesicular acidification; and that these fluxes are mediated by a K/H exchange distinct from Na/H exchange which is also present in this membrane. We conclude that a specific exchange process for K/H is located in ileal apical membrane vesicles.     

Inhibitors of Na(+)/H(+) and Na(+)/Ca(2+) exchange potentiate methamphetamine-induced dopamine neurotoxicity: possible role of ionic dysregulation in methamphetamine neurotoxicity

Although the neurotoxic potential of methamphetamine (METH) is well established, underlying mechanisms have yet to be identified. In the present study, we sought to determine whether ionic dysregulation was a feature of METH neurotoxicity. In particular, we reasoned that if METH impairs the function of Na(+)/H(+) and/or Na(+)/Ca(2+) antiporters by compromising the inward Na(+) gradient [via prolonged DA transporter (DAT) activation and Na(+)/K(+) ATPase inhibition], then amiloride (AMIL) and other inhibitors of Na(+)/H(+) and/or Na(+)/Ca(2+) exchange would potentiate METH neurotoxicity. To test this hypothesis, mice were treated with METH alone or in combination with AMIL or one of its analogs; 1 week later, the animals were killed for studies of dopamine (DA) neuronal integrity. AMIL markedly potentiated the toxic effect of METH on DA neurons. Potentiation was not caused by increased core temperature, enhanced DAT activity or higher METH brain levels. The DAT inhibitor, WIN-35,428, protected completely against METH-induced DA neurotoxicity in AMIL pretreated animals, suggesting that the potentiating effects of AMIL require a METH/DAT interaction. Findings with METH and AMIL were extended to six other AMIL analogs (MIA, EIPA, DIMA, BENZ, BEP, DiCBNZ), another species (rats), and neuronal type (5-HT neurons). These results support the notion that ionic dysregulation may play a role in METH neurotoxicity.     

A single cell model of myocardial reperfusion injury: Changes in intracellular Na+ and Ca2+ concentrations in guinea pig ventricular myocytes

To investigate the contribution of the changes in intracellular Na+ and Ca2+ concentrations ([Na+]i and [Ca2+]i) to myocardial reperfusion injury, we made an ischemia/reperfusion model in intact guinea pig myocytes. Myocardial ischemia was simulated by the perfusion of metabolic inhibitors (3.3 mM amobarbital and 5 M carbonyl cyanide m-chlorophenylhydrazone) with pH 6.6 and reperfusion was achieved by the washout of them with pH 7.4. [Na+]i increased from 7.9 ± 2.0 to 14.0 ± 3.4 mM (means ± S.E., p < 0.01) during 7.5 min of simulated ischemia (SI) and increased further to 18.8 ± 3.0 mM at 7.5 min after reperfusion. [Ca2+]i, expressed as the ratio of fluo 3 fluorescence intensity, increased to 133 ± 8% (p < 0.01) during SI and gradually returned to the control level after reperfusion. Intracellular pH decreased from 7.53 ± 0.04 to 6.31 ± 0.04 (p < 0.01) and recovered quickly after reperfusion. Reperfusion with the acidic solution or the continuous perfusion of hexamethylene amiloride (2 M) prevented the reperfusion-induced increase in [Na+]i. When the duration of SI was prolonged to 15 min, the cell response after reperfusion varied, 16 of 37 cells kept quiescent, 21 cells showed spontaneous Ca2+ waves, and 4 cells out of these 21 cells became hypercontracted. In quiescent cells, both [Na+]i and [Ca2+]i decreased immediately after reperfusion. In cells with Ca2+ waves, [Na+]i transiently increased further at the early phase of reperfusion, while [Ca+]i declined. In hypercontracted cells, [Na+]i increased as much as in Ca2+ wave cells, but [Ca2+]i increased extensively and both ion concentrations continued to increase. Reperfusion with the Ca2+-free solution prevented both the [Ca2+]i increase and morphological change. In the presence of ryanodine (10 M), the increase in [Ca2+]i after reperfusion was augmented and some cells became hypercontracted. We concluded that (1) Na+/H+ exchange is active both during SI and reperfusion, resulting in the additional [Na+]i elevation on reperfusion, (2) the [Na+]i level after reperfusion and the following Ca2+ influx via Na+/Ca2+ exchange are crucial for reperfusion cell injury, and (3) the Ca2+ buffering capacity of sarcoplasmic reticulum would also contribute to the Ca2+ regulation and cell injury after reperfusion.     

跨膜离子转运蛋白与植物耐盐的分子生物学

植物抵御盐害的主要方式是增加Na 的外排、减少Na 的吸入和Na 的区隔化,而Na 的跨膜运输主要由质膜和液泡膜上的离子转运蛋白完成。对质膜和液泡膜跨膜离子转运蛋白包括K /Na 离子转运蛋白,Na /H 逆向转运蛋白以及液泡膜H -PPase的分子生物学研究及应用进展进行了综述。     

Insulin stimulates transepithelial sodium transport by activation of a protein phosphatase that increases Na-K ATPase activity in endometrial epithelial cells.

The objective of this study was to investigate the effects of insulin and insulin-like growth factor I on transepithelial Na(+) transport across porcine glandular endometrial epithelial cells grown in primary culture. Insulin and insulin-like growth factor I acutely stimulated Na(+) transport two- to threefold by increasing Na(+)-K(+) ATPase transport activity and basolateral membrane K(+) conductance without increasing the apical membrane amiloride-sensitive Na(+) conductance. Long-term exposure to insulin for 4 d resulted in enhanced Na(+) absorption with a further increase in Na(+)-K(+) ATPase transport activity and an increase in apical membrane amiloride-sensitive Na(+) conductance. The effect of insulin on the Na(+)-K(+) ATPase was the result of an increase in V(max) for extracellular K(+) and intracellular Na(+), and an increase in affinity of the pump for Na(+). Immunohistochemical localization along with Western blot analysis of cultured porcine endometrial epithelial cells revealed the presence of alpha-1 and alpha-2 isoforms, but not the alpha-3 isoform of Na(+)-K(+) ATPase, which did not change in the presence of insulin. Insulin-stimulated Na(+) transport was inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester [HNMPA-(AM)(3)], a specific inhibitor of insulin receptor tyrosine kinase activity, suggesting that the regulation of Na(+) transport by insulin involves receptor autophosphorylation. Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase as well as okadaic acid and calyculin A, inhibitors of protein phosphatase activity, also blocked the insulin-stimulated increase in short circuit and pump currents, suggesting that activation of phosphatidylinositol 3-kinase and subsequent stimulation of a protein phosphatase mediates the action of insulin on Na(+)-K(+) ATPase activation.     

The passage of an ion through a synthetic ion channel

The simulated system consisted of a fatty acid bilayer membrane dividing two electrolyte layers each containing ions, and a channel composed of linked 15-crown-5 ether rings. The Na⁺ and F^?  ions in the aqueous electrolyte layers were too large to enter the channel, but the Li⁺ ions entered and were transported. Conditions that optimised the passive, electric-field-induced transport of Li⁺ ions through the channel were investigated. It was calculated and rationalised that the higher the numerical value of the electrostatic charge on the oxygen atoms of the crown ether rings, the more easily does the channel convey the Li⁺ ions.     

Loss of EGF binding and cation transport response during differentiation of mouse neuroblastoma cells

Mouse neuroblastoma cells (clone N1E-115) differentiate in culture upon withdrawal of serum growth factors and acquire the characteristics of neurons. We have shown tht exponentially growing N1E-115 cells possess functional epidermal growth factor (EGF) receptors but that the capacity for binding EGF and for stimulation of DNA synthesis is lost as the cells differentiate. Furthermore, in exponentially growing cells, EGF induces a rapid increase in amiloride-sensitive Na+ influx, followed by stimulation of the (Na+-K+)ATPase, indicating that activation of the Na+/H+ exchange mechanism in N1E-115 cells [1] may be induced by EGF. The ionic response is also lost during differentiation, but we have shown that the stimulation of both Na+ and K+ influx is directly proportional to the number of occupied receptors in all cells whether exponentially growing or differentiating, thus only indirectly dependent on the external EGF concentration. The linearity of the relationships indicates that there is no rate-limiting step between EGF binding and the ionic response. Our data would suggest that as neuroblastoma cells differentiate and acquire neuronal properties, their ability to respond to mitogens, both biologically and in the activation of cation transport processes, progressively decreases owing to the loss of the appropriate receptors.     

The topology of the C-terminal sections of the NCX1 Na+/Ca2+ exchanger and the NCKX2 Na+/Ca2+-K+ exchanger

Mammalian Na⁺/Ca²⁺ (NCX) and Na⁺/Ca²⁺-K⁺ exchangers (NCKX) are polytopic membrane proteins that play critical roles in calcium homeostasis in many cells. Although hydropathy plots for NCX and NCKX are very similar, reported topological models for NCX1 and NCKX2 differ in the orientation of the three C-terminal transmembrane segments (TMS). NCX1 is thought to have 9 TMS and a re-entrant loop, whereas NCKX2 is thought to have 10 TMS. The current topological model of NCKX2 is very similar to the 10 membrane spanning helices seen in the recently reported crystal structure of NCX_MJ, a distantly related archaebacterial Na⁺/Ca²⁺ exchanger. Here we reinvestigate the orientation of the three C-terminal TMS of NCX1 and NCKX2 using mass-tagging experiments of substituted cysteine residues. Our results suggest that NCX1, NCKX2 and NCX_MJ all share the same 10 TMS topology.     

The co-presence of Na+/Ca2+-K+ exchanger and Na+/Ca2+ exchanger in bovine adrenal chromaffin cells

We have previously shown that there is high Na(+)/Ca(2+) exchange (NCX) activity in bovine adrenal chromaffin cells. In this study, by monitoring the [Ca(2+)](i) change in single cells and in a population of chromaffin cells, when the reverse mode of exchanger activity has been initiated, we have shown that the NCX activity is enhanced by K(+). The K(+)-enhanced activity accounted for a significant proportion of the Na(+)-dependent Ca(2+) uptake activity in the chromaffin cells. The results support the hypothesis that both NCX and Na(+)/Ca(2+)-K(+) exchanger (NCKX) are co-present in chromaffin cells. The expression of NCKX in chromaffin cells was further confirmed using PCR and northern blotting. In addition to the plasma membrane, the exchanger activity, measured by Na(+)-dependent (45)Ca(2+) uptake, was also present in membrane isolated from the chromaffin granules enriched fraction and the mitochondria enriched fraction. The results support that both NCX and NCKX are present in bovine chromaffin cells and that the regulation of [Ca(2+)](i) is probably more efficient with the participation of NCKX.     

Alkali metal ion selectivity of the hemocyanin channel

Summary The selectivity of the hemocyanin channel was measured for alkali metal ions and ammonium. Permeability ratios relative to K⁺ measured from biionic potentials were: NH ₄ ⁺ (1.52)>Rb⁺ (1.05)>K⁺ (1.0)>Cs⁺ (0.89)>Na⁺ (0.81)>Li⁺ (0.35). Single-channel ion conductance was a saturating function of ion concentration regardless of the cation present in the bathing medium. Maximal conductances were 270, 267, 215, 176, 170 and 37 ps for K⁺, Rb⁺, NH ₄ ⁺ , Cs⁺, Na⁺ and Li⁺, respectively. Current-voltage curves for the different monovalent cations were measured and described using a threebarrier model previously used to explain the voltage dependence of the instantaneous channel conductance (Cecchi, Alvarez & Latorre, 1981). In this way, binding and peak energies were estimated for the different ions. Considering the energy peaks as transition states between the ion and the channel, it is concluded that they follow Eisenman's selectivity sequences XI (cis peak, i.e., Li⁺>Na⁺>K⁺>Rb⁺>Cs⁺; highest field strength), VII (central peak) and II (trans peak). The cis side was that to which hemocyanin was added and was electrically ground. The binding energies, on the other hand, follow Eisenman's series XI for strong electric field sites. Binding of NH ₄ ⁺ to the cis-well suggests that the orientation of the ligands in the site is tetrahedric.     

Iron-Induced Inhibition of Na+, K+-ATPase and Na+/Ca2+ Exchanger in Synaptosomes: Protection by the Pyridoindole Stobadine

The effect of oxidative stress, induced by Fe²⁺-EDTA system, on Na⁺,K⁺-ATPase, Na⁺/Ca²⁺ exchanger and membrane fluidity of synaptosomes was investigated. Synaptosomes isolated from gerbil whole forebrain were incubated in the presence of 200 M FeSO₄-EDTA per mg of protein at 37°C for 30 min. The oxidative insult reduced Na⁺,K⁺-ATPase activity by 50.7 ± 5.0 % and Na⁺/Ca²⁺ exchanger activity measured in potassium and choline media by 47.1 ± 7.2 % and 46.7 ± 8.6 %, respectively. Membrane fluidity was also significantly reduced as observed with the 1,6-diphenyl-1,3,5-hexatriene probe. Stobadine, a pyridoindole derivative, prevented the decrease in membrane fluidity and in Na⁺/Ca²⁺ exchanger activity. The Na⁺,K⁺-ATPase activity was only partially protected by this lipid antioxidant, indicating a more complex mechanism of inhibition of this protein. The results of the present study suggest that the Na⁺/Ca²⁺ exchanger and the Na⁺,K⁺-ATPase are involved in oxidation stress-mediated disturbances of intracellular ion homeostasis and may contribute to cell injury.     

Changes in ion content and transport during development of embryonic rainbow trout

Wet mass and water content of four lots of whole eggs did not change throughout embryonic development of rainbow trout Oncorhynchus mykiss. Eggs in all four lots accumulated Na⁺. Eggs in lots 2 and 4 also accumulated Ca²⁺ and Cl^-, whereas eggs in lot 1 showed no significant change in Ca²⁺ or Cl^- and eggs in lot 3 showed no change in Cl^-and a small loss of Ca²⁺. Although the Na⁺ content of embryonic tissues increases in the later stages of development, the yolk sac content remained constant, indicating uptake of Na⁺ from the environment. Na+ uptake by whole eggs was non-saturable, consistent with diffusion of Na⁺ across the chorion into the perivitelline fluid. Na⁺ uptake in dechorionated embryos was saturable, as was Ca²⁺ uptake by both whole eggs and dechorionated embryos, consistent with active uptake or facilitated diffusion mechanisms at the surface of embryos. Very low Ca²⁺ uptake rates in dechorionated embryos suggest that the Ca²⁺ uptake mechanism is not fully developed until after hatching.