Peritubular membrane potential in kidney proximal tubular cells of spontaneously hypertensive rats

Peritubular membrane potential in kidney proximal tubular cells of spontaneously hypertensive rats (SHR-Okamoto strain adult rats) was measured with conventional 3 mol KCl microelectrodes, in vivo. Peritubular cell membrane potential was not different in SHR (-66.5 ± 0.7 mV) as compared with normotensive control Wistar rats (-67.5 ± 1.2 mV). To test the effects of possible altered sodium membrane transport in SHR on proximal tubule peritubular membrane potential, we allowed SHR and control rats to drink 1% NaCl for two weeks. Again, proximal tubule peritubular membrane potential was not different in SHR on 1% NaCl (-67.0 ± 1.0 mV) as compared with control rats on 1% NaCl (-64.7 ± 1.3 mV). From these results we concluded that peritubular membrane potential in kidney proximal tubular cells of SHR was not different from normotensive Wistar control rats, and if some alteration of sodium transport in kidney proximal tubular cells of SHR could exist, that was not possible to evaluate from the measurements of peritubular membrane potential in kidney proximal tubular cells.     

Effects of Ce3+, Cd2+, and Hg2+ on activities and secondary structure of trypsin

The different effects of Ce³⁺, Cd²⁺, and Hg²⁺ on the activities and secondary structure of trypsin were studied. The results showed that trypsin activity was increased substantially by Ce³⁺ in 0.5–5 μmol/L concentration, but the activity was decreased significantly by Cd²⁺ or Hg²⁺ in 0.5–5 μmol/L concentration. The ultraviolet-visible spectrum of trypsin with 4 μmol/L Ce³⁺ treatment was the same as that of the control, but the 232-nm characteristic peak of trypsin with 4 μmol/L Cd²⁺ or Hg²⁺ treatment was blue-shifted and the peak intensity weakened. The circular dichroism (CD) spectrum of trypsin with 4 μmol/L Ce³⁺ treatment was similar to that of the control. The secondary structure of trypsin did not change with Ce³⁺ treatment. However, the CD spectrum of trypsin with 4 μmol/L Cd²⁺ or Hg²⁺ treatment was different from that of the control and Ce³⁺ treatment. The secondary structure of trypsin with Cd²⁺ or Hg²⁺ treatment changed greatly; for example, the α-helix and β-sheet contents were reduced significantly, the β-turn was enhanced greatly, and the random coil contents increased or decreased.     

Basolateral membrane Na(+)-independent Cl-/HCO3- exchange in the inner stripe of the rabbit outer medullary collecting tubule

The inner stripe of the outer medullary collecting tubule is a major distal nephron segment in urinary acidification. To examine the mechanism of basolateral membrane H+/OH-/HCO3- transport in this segment, cell pH was measured microfluorometrically in the inner stripe of the rabbit outer medullary collecting tubule perfused in vitro using the pH-sensitive fluorescent dye, (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein. Decreasing peritubular pH from 7.4 to 6.8 (changing [HCO3-] from 25 to 5 mM) caused a cell acidification of 0.25 +/- 0.02 pH units, while a similar luminal change resulted in a smaller cell acidification of only 0.04 +/- 0.01 pH units. Total replacement of peritubular Cl- with gluconate caused cell pH to increase by 0.18 +/- 0.04 pH units, an effect inhibited by 100 microM peritubular DIDS and independent of Na+. Direct coupling between Cl- and base was suggested by the continued presence of peritubular Cl- removal-induced cell alkalinization under the condition of a cell voltage clamp (K(+)-valinomycin). In addition, 90% of basolateral membrane H+/OH-/HCO3- permeability was inhibited by complete removal of luminal and peritubular Cl-. Peritubular Cl(-)-induced cell pH changes were inhibited two-thirds by removal of exogenous CO2/HCO3- from the system. The apparent Km for peritubular Cl- determined in the presence of 25 mM luminal and peritubular [HCO3-] was 113.5 +/- 14.8 mM. These results demonstrate that the basolateral membrane of the inner stripe of the outer medullary collecting tubule possesses a stilbene-sensitive Cl-/HCO3- exchanger which mediates 90% of basolateral membrane H+/OH-/HCO3- permeability and may be regulated by physiologic Cl- concentrations.     

Enhancement of HERG K(+) currents by Cd(2+) destabilization of the inactivated state

We have studied the functional effects of extracellular Cd(2+) on human ether-a-go-go-related gene (HERG) encoded K(+) channels. Low concentrations (10-200 &mgr;M) of extracellular Cd(2+) increased outward currents through HERG channels; 200 &mgr;M Cd(2+) more than doubled HERG currents and altered current kinetics. Cd(2+) concentrations up to 200 &mgr;M did not change the voltage dependence of channel activation, but shifted the voltage dependence of inactivation to more depolarized membrane potentials. Cd(2+) concentrations >/=500 &mgr;M shifted the voltage dependence of channel activation to more positive potentials. These results are consistent with a somewhat specific ability of Cd(2+) to destabilize the inactivated state. We tested the hypothesis that channel inactivation is essential for Cd(2+)-induced increases in HERG K(+) currents, using a double point mutation (G628C/S631C) that diminishes HERG inactivation (Smith, P. L., T. Baukrowitz, and G. Yellen. 1996. Nature (Lond.). 379:833-836). This inactivation-removed mutant is insensitive to low concentrations of Cd(2+). Thus, Cd(2+) had two distinct effects on HERG K(+) channels. Low concentrations of Cd(2+) caused relatively selective effects on inactivation, resulting in a reduction of the apparent rectification of the channel and thereby increasing HERG K(+) currents. Higher Cd(2+) concentrations affected activation gating as well, possibly by a surface charge screening mechanism or by association with a lower affinity site.     

Kinetics of Na(+) transport in necturus proximal tubule

The dependence of proximal tubular sodium and fluid readsorption on the Na(+) concentration of the luminal and peritubular fluid was studied in the perfused necturus kidney. Fluid droplets, separated by oil from the tubular contents and identical in composition to the vascular perfusate, were introduced into proximal tubules, reaspirated, and analyzed for Na(+) and [(14)C]mannitol. In addition, fluid transport was measured in short-circuited fluid samples by observing the rate of change in length of the split droplets in the tubular lumen. Both reabsorptive fluid and calculated Na fluxes were simple, storable functions of the perfusate Na(+) concentration (K(m) = 35-39 mM/liter, V(max) = 1.37 control value). Intracellular Na(+), determined by tissue analysis, and open-circuit transepithelial electrical potential differences were also saturable functions of extracellular Na(+). In contrast, net reabsorptive fluid and Na(+) fluxes were linearly dependent on intracellular Na(+) and showed no saturation, even at sharply elevated cellular sodium concentrations. These concentrations were achieved by addition of amphotericin B to the luminal perfusate, a maneuver which increased the rate of Na(+) entry into the tubule cells and caused a proportionate rise in net Na(+) flux. It is concluded that active peritubular sodium transport in proximal tubule cells of necturus is normally unsaturated and remains so even after amphotericin-induced enhancement of luminal Na(+) entry. Transepithelial movement of NaCl may be described by a model with a saturable luminal entry step of Na(+) or NaCl into the cell and a second, unsaturated active transport step of Na(+) across the peritubular cell boundary.     

Organic osmolytes betaine,sorbitol and inositol are potent inhibitors of erythrocyte membrane ATPases

Organic osmolytes are used in animal and plant cells to adapt to hyper- and hypoosmolar stress. We used our RBC-membrane model to investigate the effects of the osmolytes betaine, sorbitol and myo-inositol on Na(+)/K(+)-ATPase, Ca(2+)-ATPase and calmodulin-stimulated Ca(2+)-ATPase (CaM). Our results show that betaine inhibited ATPases by more than 61%: Na(+)/K(+)-ATPase (75 +/- 5.9 vs 27 +/- 2.2), Ca(2+)-ATPase (236 +/- 18.9 vs 62 +/- 4.9), and CaM (450 +/- 18 vs 174 +/- 6.9) (microM pi/min/mg protein, control (0 microM betaine) vs 100 micromol/L betaine). Sorbitol (100 micromol/L) inhibited the Ca(2+)-ATPases by 41% (126 +/- 7.6 vs 74 +/- 4.4) and CaM by 42% (253 +/- 17.7 vs 147 +/- 10.3). Inositol (100 micromol/L) inhibited Na(+)/K(+)-ATPase strongest (37 +/- 1.9 vs 20 +/- 1.0; 47% inhibition) while it showed a lesser effect on the Ca(2+)-ATPases (136 +/- 6.8 vs 102 +/- 5.1; 25% inhibition). All osmolytes inhibited RBC membrane ATPases at concentrations above 50 micromol/L, which corresponds to high normal physiologic range for organic osmolytes in serum. Furthermore, the presence of osmolytes (250 micromol/L) decreased hypoosmotic stress induced hemolysis by 42%. Together these data indicate an important regulatory role of organic osmolytes on human RBC membrane ATPases and a protective function of osmolytes in RBCs against hypoosmotic stress.     

Absence of KCNQ1-dependent K+ fluxes in proximal tubular cells of frog kidney

The present study was designed to investigate the functional significance of KCNQ1-mediated K+ secretory fluxes in proximal tubular cells of the frog kidney. To this end, we investigated the effects on rapid depolarization and slow repolarization of the peritubular membrane potential after luminal addition of L-phenylalanine or L-alanine plus/minus KCNQ1 channel blockers. Perfusing the lumen with 10 mmol/L L-phenylalanine plus/minus luminal 293B, a specific blocker of KCNQ1, did not modify the rapid depolarization and the rate of slow repolarization. Perfusing the lumen with 10 mmol/L L-alanine plus/minus luminal HMR-1556, a more potent KCNQ1 channel blocker, did not also alter the rapid depolarization and the rate of slow repolarization. Pretreatment (1 h) of the lumen with HMR-1556 also failed to modify rapid depolarization and rate of slow repolarization upon luminal 10 mmol/L L-alanine. Perfusing the lumen with 1 mmol/L L-alanine plus/minus luminal HMR-1556 did not change the rapid depolarization and the rate of slow repolarization. The pretreatment (1 h) with luminal HMR-1556 did not modify the rapid depolarization and the rate of slow repolarization upon luminal 1 mmol/L L-alanine. The pretreatment (1 h) of the lumen with HMR-1556 did not change transference number for K+ of peritubular cell membrane. Finally, luminal barium blunted the rapid depolarization upon application of luminal 1 mmol/L L-alanine. RT-PCR showed that KCNQ1 mRNA was not expressed in frog kidney. In conclusion, the KCNQ1-dependent K+ secretory fluxes are absent in proximal tubule of frog kidney.     

Silver ions disrupt K⁺ homeostasis and cellular integrity in intact barley (Hordeum vulgare L.) roots

The heavy metals silver, gold, and mercury can strongly inhibit aquaporin-mediated water flow across plant cell membranes, but critical examinations of their side effects are rare. Here, the short-lived radiotracer (42)K is used to demonstrate that these metals, especially silver, profoundly change potassium homeostasis in roots of intact barley (Hordeum vulgare L.) plants, by altering unidirectional K(+) fluxes. Doses as low as 5 μM AgNO(3) rapidly reduced K(+) influx to 5% that of controls, and brought about pronounced and immediate increases in K(+) efflux, while higher doses of Au(3+) and Hg(2+) were required to produce similar responses. Reduced influx and enhanced efflux of K(+) resulted in a net loss of >40% of root tissue K(+) during a 15 min application of 500 μM AgNO(3), comprising the entire cytosolic potassium pool and about a third of the vacuolar pool. Silver also brought about major losses of UV-absorbing compounds, total electrolytes, and NH(4)(+). Co-application, with silver, of the channel blockers Cs(+), TEA(+), or Ca(2+), did not affect the enhanced efflux, ruling out the involvement of outwardly rectifying ion channels. Taken together with an examination of propidium iodide staining under confocal microscopy, the results indicate that silver ions affect K(+) homeostasis by directly inhibiting K(+) influx at lower concentrations, and indirectly inhibiting K(+) influx and enhancing K(+) efflux, via membrane destruction, at higher concentrations. Ni(2+), Cd(2+), and Pb(2+), three heavy metals not generally known to affect aquaporins, did not enhance K(+) efflux or cause propidium iodide incorporation. The study reveals strong and previously unknown effects of major aquaporin inhibitors and recommends caution in their application.     

Effects of changes in sodium electrochemical potential gradient on p-aminohippurate transport in newt kidney

1. The relation between p-aminohippurate uptake and the electrochemical potential gradient of Na+ (delta muNa+) across the peritubular membrane was examined in newt (Triturus pyrrhogaster) kidney. The delta muNa+ was modified by changing cellular Na+ concentration and/or lowering the electrical potential difference across the peritubular membrane (peritubular membrane potential) 2. Elevation of external K+ concentration or addition of alanine at 40 mM to the medium decreased the delta muNa+ mainly through the depolarization of the cells. Addition of 1 mM ouabain resulted in a decrease in the peritubular membrane potential and increase in cellular Na+ concentration, thus decrease in the delta muNa+. 3. p-Aminohippurate uptake decreased in proportion to the decrease in the delta muNa+ under all experimental conditions, indicating that the maintenance of the delta muNa+ is required for p-aminohippurate transport. 4. All three different experimental conditions, high medium K+ concentration, 40 mM alanine or 1 mM ouabain, increased the apparent Michaelis constant, Kt, without affecting the maximal uptake rate, V, for p-aminohippurate. These results suggests that the delta muNa+, largely the peritubular membrane potential, may affect the association and/or dissociation of p-aminohippurate and Na+ at both interfaces of the peritubular membrane of the proximal tubular cells.     

Kinetic transport model for cellular regulation of pH and solute concentration in the renal proximal tubule.

An open circuit kinetic model was developed to calculate the time course of proximal tubule cell pH, solute concentrations, and volume in response to induced perturbations in luminal or peritubular fluid composition. Solute fluxes were calculated from electrokinetic equations containing terms for known carrier saturabilities, allosteric dependences, and ion coupling ratios. Apical and basolateral membrane potentials were determined iteratively from the requirements of cell electroneutrality and equal opposing transcellular and paracellular currents. The model converged to membrane potentials accurate to 0.05% in one to four iterations. Model variables included cell concentrations of Na, K, HCO3, glucose, pH (uniform CO2), volume, and apical and basolateral membrane potentials. The basic model contained passive apical membrane transport of Na/H, Na/glucose, H and K, basolateral transport of Na/3HCO3, K, H, and glucose, and paracellular transport of Na, K, Cl, and HCO3; apical H and basolateral 3Na/2K-ATPases were present. Apical Na/H and basolateral K transport were regulated allosterically by pH. Apical Na/H transport, basolateral Na/3HCO3 transport, and the 3Na/2K-ATPase were saturable. Model parameters were chosen from data in the rat proximal tubule. Model predictions for the magnitude and time course of cell pH, Na, and membrane potential in response to rapid changes in apical and peritubular Na and HCO3 were in excellent agreement with experiment. In addition, the model requires that there exist an apical H-ATPase, basolateral Na/3HCO3 transport saturable with HCO3, and electroneutral basolateral K transport.     

Cadmium-mediated oxidative stress in kidney proximal tubule cells induces degradation of Na+/K(+)-ATPase through proteasomal and endo-/lysosomal proteolytic pathways.

The mechanisms of cadmium (Cd)-dependent nephrotoxicity were studied in a rat proximal tubule (PT) cell line. CdCl(2) (5 microM) increased the production of reactive oxygen species (ROS), as determined by oxidation of dihydrorhodamine 123 to fluorescent rhodamine 123. The levels of ubiquitin-conjugated cellular proteins were increased by Cd in a time-dependent fashion (maximum at 24-48 h). This was prevented by coincubation with the thiol antioxidant N-acetylcysteine (NAC, 15 mM). Cd also increased apoptosis (controls: 2.4+/-1.6%; Cd: 8.1+/-1.9%), but not necrosis (controls: 0.5 +/- 0.3%; Cd: 1.4+/- 2.5%). Exposure of PT cells with Cd decreased protein levels of the catalytic subunit (alpha1) of Na+/K(+)-ATPase, a long-lived membrane protein (t(1/2)>48 h) that drives reabsorption of ions and nutrients through Na(+)-dependent transporters in PT. Incubation of PT cells for 48 h with Cd decreased Na+/K(+)-ATPase alpha1-subunit, as determined by immunoblotting, by approximately 50%, and NAC largely prevented this effect. Inhibitors of the proteasome such as MG-132 (20 microM) or lactacystin (10 microM), as well as lysosomotropic weak bases such as chloroquine (0.2 mM) or NH(4)Cl (30 mM), significantly reduced the decrease of Na(+)/K(+)-ATPase alpha1-subunit induced by Cd, and in combination abolished the effect of Cd on Na+/K(+)-ATPase. Immunofluorescence labeling of Na+/K(+)-ATPase showed a reduced expression of the protein in the plasma membrane of Cd-exposed cells. After addition of lactacystin and chloroquine to Cd-exposed PT cells, immunoreactive material accumulated into intracellular vesicles. The data indicate that micromolar concentrations of Cd can increase ROS production and exert a toxic effect on PT cells. Oxidative damage increases the degradation of Na+/K(+)-ATPase through both the proteasomal and endo-/lysosomal proteolytic pathways. Degradation of oxidatively damaged Na+/K(+)-ATPase may contribute to the 'Fanconi syndrome'-like Na(+)-dependent transport defects associated with Cd-nephrotoxicity.     

Effects of cumene hydroperoxide on cellular cation composition in frog kidney proximal tubular cells

Effects of cumene hydroperoxide were studied on the peritubular membrane potential and cellular cation composition in frog kidney proximal tubular cells. After perfusion of isolated frog kidneys for 30 min with 1.3x10(-4) mol l(-1) cumene hydroperoxide Ringer solution, the peritubular membrane potential gradually declined. The ouabain-like effects were demonstrated on cell Na and K activities after 1 h of perfusion with cumene hydroperoxide. The peritubular apparent transference number for potassium was decreased. Intracellular pH was not altered in the presence of cumene hydroperoxide. Intracellular free Ca(2+) concentration increased slowly and moderately. The concentration of the malondialdehyde in the kidney homogenates, measured as an index of lipid peroxidation, was increased. A previously observable effect of cumene hydroperoxide on the peritubular membrane potential was prevented by oxygen radical scavengers.     

电压激活的钾通道阻断剂抑制山莨菪碱松弛去甲肾上腺素预收缩的兔主动脉平滑肌

研究显示,山莨菪碱预处理不改变高钾引起的兔主动脉环收缩,但可明显减弱去甲肾上腺素(noradrenaline,NA)、组织胺或5-羟色胺引起的收缩,且其减弱作用不受去除血管内皮影响。本实验观察了几种钾通道阻断剂对山良菪碱松弛：NA预收缩的兔主动脉环的影响。结果表明,1、3、10μmol／L山莨菪碱作用8min,可使0．01μmol／L NA预收缩的兔主动脉环松弛(P＜O．01)。10mmol／L,CsCl、1mmol／L 4-氨基吡啶、10μmol／L BaCl2、10μmol/L格列本脲、3μmol／L charybdotoxin和3μmol／L蜂毒明从分别与0．0lμmol／L NA同时加入,可增强后者收缩兔主动脉环的作用(P＜0．01)。10、30mmol／L CsCl或10、30mmol／L 4-氨基吡啶存在时,10μmol／L山茛菪碱对NA预收缩的兔主动脉环的松弛作用减弱,松弛率与对照组比较分别有极显著差异(P＜0．01);10、30μmol／L BaCl2,10、30μmol/L格列本脲,3μmol/L charybdotoxin或3μmol／L蜂毒明肽存在时,山莨菪碱对NA预收缩的兔主动脉环的松弛作用不受影响(P＞O．05)。本研究表明,电压激活的钾通道阻断剂抑制山莨菪碱松弛NA预收缩的兔主动脉平滑肌,初步提示血管平滑肌细胞膜上电压激活的钾通道参与山莨菪碱扩血管作用。     

A type of voltage-dependent Ca2+ channel on Vicia faba guard cell plasma membrane outwardly permeates K+

The fine regulation of stomatal aperture is important for both plant photosynthesis and transpiration, while stomatal closing is an essential plant response to biotic and abiotic stresses such as drought, salinity, wounding, and pathogens. Quick stomatal closing is primarily due to rapid solute loss. Cytosolic free calcium ([Ca(2+)](cyt)) is a ubiquitous second messenger, and its elevation or oscillation plays important roles in stomatal movements, which can be triggered by the opening of Ca(2+)-permeable channels on the plasma membrane. For Ca(2+)-permeable channel recordings, Ba(2+) is preferred as a charge-carrying ion because it has higher permeability to Ca(2+) channels and blocks K(+) channel activities to facilitate current recordings; however, it prevents visualization of Ca(2+) channels' K(+) permeability. Here, we employed Ca(2+) instead of Ba(2+) in recording Ca(2+)-permeable channels on Vicia faba guard cell plasma membrane to mimic physiological solute conditions inside guard cells more accurately. Inward Ca(2+) currents could be recorded at the single-channel level, and these currents could be inhibited by micromolar Gd(3+), but their reversal potential is far away from the theoretical equilibrium potential for Ca(2+). Further experiments showed that the discrepancy of the reversal potential of the recorded Ca(2+) currents is influenced by cytosolic K(+). This suggests that voltage-dependent Ca(2+) channels also mediate K(+) efflux at depolarization voltages. In addition, a new kind of high-conductance channels with fivefold to normal Ca(2+) channel and 18-fold to normal outward K(+) conductance was found. Our data presented here suggest that plants have their own saving strategies in their rapid response to stress stimuli, and multiple kinds of hyperpolarization-activated Ca(2+)-permeable channels coexist on plasma membranes.     

The measurement of intracellular sodium activities in the bullfrog by means of double-barreled sodium liquid ion-exchanger microelectrodes.

A double-barreled Na+-selective microelectrode was constructed with monensin as a liquid ion exchanger. The HCl-treated monensin was dissolved in a solvent (Corning 477317) at 10% (weight/weight). Internal reference solution of its ionic barrel was mixture of 0.49 M NaCl and 0.01 M KCl, the pH being adjusted to 3 with 0.1 M citrate-HCl buffer, whereas that of the PD barrel was 0.5 M KCl. Average slope and selectivity ratio (Na+/K+) tested on 10 different microelectrodes were -57.5 +/- 1.87 mV/P(Na) (SEM) and 6.7 +/- 0.44, respectively. The electrical resistance was an order of 10(10) ohm and the response time was less than 10 sec. Using this microelectrode, a free flow micropuncture experiment was carried out in the bullfrog kidney and the intracellular Na+ activity as well as the membrane PD was determined on the proximal tubular cell. Average value (+/- SEM, n = 15) for the intracellular Na+ and K+ was 20.7 +/- 1.56 mEq/L and 61.2 +/- 1.16 mEq/L, respectively, and -68.7 +/- 0.88 mV for the peritubular membrane PD. There was a significant negative correlation between Na+ and K+ activities within the cell, i.e., the lower the ionic activity of cellular Na+ was, the higher the cellular K+, and vice versa, the sum of these two being kept nearly constant. The above finding may be somehow related to the isosmosis in the reabsorptive process across the proximal tubular epithelium.     

Effect of Cd(2+) and Hg(2+) on the activity of Na(+)/K(+)-ATPase and Mg(2+)-ATPase adsorbed on polystyrene microtiter plates.

In the present study a polystyrene microtiter plate was tested as a support material for synaptic plasma membrane (SPM) immobilization by adsorption. The adsorption was carried out by an 18-h incubation at +4 degrees C of SPM with a polystyrene matrix, at pH 7.4. Evaluation of the efficiency of the applied immobilization method revealed that 10% protein fraction of initially applied SPM was bound to the support and that two SPM enzymes, Na(+)/K(+)-ATPase and Mg(2+)-ATPase, retained 70-80% activity after the adsorption. In addition, adsorption stabilizes Na(+)/K(+)-ATPase and Mg(2+)-ATPase, since the activities are substantial 3 weeks after the adsorption. Parallel kinetic analysis showed that adsorption does not alter significantly the kinetic properties of Na(+)/K(+)-ATPase and Mg(2+)-ATPase and their sensitivity to and mechanism of Cd(2+)- or Hg(2+)-induced inhibition. The only exception is the "high affinity" Mg(2+)-ATPase moiety, whose affinity for ATP and sensitivity toward Cd(2+) were increased by the adsorption. The results show that such system may be used as a practical and comfortable model for the in vitro toxicological investigations.     

Characterization of Na/Ca exchange in plasmalemmal vesicles from zona fasciculata cells of the bovine adrenal gland

The presence of an Na/Ca exchange system in fasciculata cells of the bovine adrenal gland was tested using isolated plasmalemmal vesicles. In the presence of an outwardly Na(+) gradient, Ca(2+) uptake was about 2-fold higher than in K(+) condition. Li(+) did not substitute for Na(+) and 5 mM Ni(2+) inhibited Ca(2+) uptake. Ca(2+) efflux from Ca(2+)-loaded vesicles was Na(+)-stimulated and Ni(2+)-inhibited. The saturable part of Na(+)-dependent Ca(2+) uptake displayed Michaelis-Menten kinetics. The relationship of Na(+)-dependent Ca(2+) uptake versus intravesicular Na(+) concentration was sigmoid (apparent K(0.5) approximately 24 mM; Hill number approximately 3) and Na(+) acted on V(max) without significant effect on K(m). Na(+)-stimulated Ca(2+) uptake was temperature-dependent (apparent Q(10) approximately 2.2). The inhibition properties of several divalent cations (Cd(2+), Sr(2+), Ni(2+), Ba(2+), Mn(2+), Mg(2+)) were tested and were similar to those observed in kidney basolateral membrane. The above results indicate the presence of an Na/Ca exchanger located on plasma membrane of zona fasciculata cells of bovine adrenal gland. This exchanger displays similarities with that of renal basolateral cell membrane.     

Mechanism of basolateral membrane H+/OH-/HCO-3 transport in the rat proximal convoluted tubule. A sodium-coupled electrogenic process

In order to examine the mechanism of basolateral membrane H+/OH-/HCO-3 transport, a method was developed for the measurement of cell pH in the vivo doubly microperfused rat proximal convoluted tubule. A pH-sensitive fluorescein derivative, (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein, was loaded into cells and relative changes in fluorescence at two excitation wavelengths were followed. Calibration was accomplished using nigericin with high extracellular potassium concentrations. When luminal and peritubular fluids were pH 7.32, cell pH was 7.14 +/- 0.01. Decreasing peritubular pH from 7.32 to 6.63 caused cell pH to decrease from 7.16 +/- 0.02 to 6.90 +/- 0.03. This effect occurred at an initial rate of 2.4 +/- 0.3 pH units/min, and was inhibited by 0.5 mM SITS. Lowering the peritubular sodium concentration from 147 to 25 meq/liter caused cell pH to decrease from 7.20 +/- 0.03 to 6.99 +/- 0.01. The effect of peritubular sodium concentration on cell pH was inhibited by 0.5 mM SITS, but was unaffected by 1 mM amiloride. In addition, when peritubular pH was decreased in the total absence of luminal and peritubular sodium, the rate of cell acidification was 0.2 +/- 0.1 pH units/min, a greater than 90% decrease from that in the presence of sodium. Cell depolarization achieved by increasing the peritubular potassium concentration caused cell pH to increase, an effect that was blocked by peritubular barium or luminal and peritubular sodium removal. Lowering the peritubular chloride concentration from 128 to 0 meq/liter did not affect cell pH. These results suggest the existence of an electrogenic, sodium-coupled H+/OH-/HCO-3 transport mechanism on the basolateral membrane of the rat proximal convoluted tubule.     

Inhibition of Na+/K(+)-ATPase and Mg(2+)-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na(+)/K(+)-ATPase and Mg(2+)-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu(2+), Zn(2+), Fe(2+) and Co(2+)) and heavy metals (Hg(2+) and Pb(2+)) ions were studied. All investigated metals produced a larger maximum inhibition of Na(+)/K(+)-ATPase than Mg(2+)-ATPase activity. The free concentrations of the key species (inhibitor, MgATP(2-), MeATP(2-)) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu(2+)/Fe(2+) or Hg(2+)/Pb(2+) caused additive inhibition, while Cu(2+)/Zn(2+) or Fe(2+)/Zn(2+) inhibited Na(+)/K(+)-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu(2+)/Fe(2+) or Cu(2+)/Zn(2+) inhibited Mg(2+)-ATPase activity synergistically, while Hg(2+)/Pb(2+) or Fe(2+)/Zn(2+) induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na(+)/K(+)-ATPase activity by reducing the maximum velocities (V(max)) rather than the apparent affinity (Km) for substrate MgATP(2-), implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg(2+)-ATPase activity by Zn(2+), Fe(2+) and Co(2+) as well as kinetic analysis indicated two distinct Mg(2+)-ATPase subtypes activated in the presence of low and high MgATP(2-) concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na(+)/K(+)-ATPase with various potencies. Furthermore, these ligands also reversed Na(+)/K(+)-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg(2+)-induced inhibition was not obtained.