Extracellular Protons Regulate the Extracellular Cation Selectivity of the Sodium Pump

The effects of 0.3–10 nM extracellular protons (pH 9.5–8.0) on ouabain-sensitive rubidium influx were determined in 4,4′-diisocyanostilbene-2, 2′-disulfonate (DIDS)-treated human and rat erythrocytes. This treatment clamps the intracellular H. We found that rubidium binds much better to the protonated pump than the unprotonated pump; 13-fold better in rat and 34-fold better in human erythrocytes. This clearly shows that protons are not competing with rubidium in this proton concentration range. Bretylium and tetrapropylammonium also bind much better to the protonated pump than the unprotonated pump in human erythrocytes and in this sense they are potassium-like ions. In contrast, guanidinium and sodium bind about equally well to protonated and unprotonated pump in human red cells. In rat red cells, protons actually make sodium bind less well (about sevenfold). Thus, protons have substantially different effects on the binding of rubidium and sodium. The effect of protons on ouabain binding in rat red cells was intermediate between the effects of protons on rubidium binding and on sodium binding. Remarkably, all four cationic inhibitors (bretylium, guanidinium, sodium, and tetrapropylammonium) had similar apparent inhibitory constants for the unprotonated pump (∼5–10 mM). The Kd for proton binding to the human pump, with the empty transport site facing extracellularly is 13 nM, whereas the extracellular transport site loaded with sodium is 9.5 nM, and with rubidium is 0.38 nM. In rat red cells there is also a substantial difference in the Kd for proton binding to the sodium-loaded pump (14.5 nM) and the rubidium-loaded pump (0.158 nM). These data suggest that important rearrangements occur at the extracellular pump surface as the pump moves between conformations in which the outward facing transport site has sodium bound, is empty, or has rubidium bound and that guanidinium is sodium-like and bretylium and tetrapropylammonium are rubidium-like.

Cystine dimethyl ester reduces the forces driving sodium-dependent transport in LLC-PK1 cells

Cystinosis is an inherited metabolic disease characterized by accumulation of lysosomal cystine and renal impairment. In an attempt to better understand the link between cystine accumulation and renal functions, we studied the effects of cystine loading on the Na(+)-H+ antiporter and the sodium pump in renal epithelial cells (LLC-PK1) in culture. Incubation of LLC-PK1 with 1 mM cystine dimethyl ester (CDME) for 48 h caused lysosomal cystine loading and reduced by 22 +/- 2% the maximal velocity of sodium-hydrogen antiport with no significant change in the affinity of sodium for the transporter. Rubidium influx decreased to 46 +/- 5% of control. Ouabain binding experiments revealed a 10% reduction in the number of Na(+)-K(+)-ATPase units in the intact cells. Na(+)-K(+)-ATPase activity in the particulate fraction of the cells homogenate declined to 50 +/- 7.5% of controls. No significant change was observed in the activity of ouabain-insensitive phosphatases. The intracellular concentration of sodium increased from 20.6 +/- 3.7 to 64.8 +/- 10 mM, and potassium concentration decreased from 103 +/- 6 to 80 +/- 13 mM. In addition to the observed reduction in the sodium gradient and in agreement with the reduction in the intracellular potassium concentration, the membrane potential changed from -80.8 +/- 7.5 to -69.9 +/- 7.0 mV. The results suggest that intracellular accumulation of cystine is associated with reduction in the number and the activity of membrane transporters. The consequence of the changes in the activity of Na(+)-K(+)-ATPase is a reduction in the electrochemical forces that drive transport in the renal cells tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of Na-K-ATPase-mediated rubidium influx in single segments of rat nephron

To determine the functioning rate of Na-K-ATPase in the rat nephron, a micromethod was developed to measure the rate of rubidium uptake in single nephron segments microdissected from collagenase-treated kidneys. Because the hydrolytic activity of Na-K-ATPase displayed the same apparent affinity for K and Rb ions, whereas the Vmax elicited by K was higher than that in the presence of Rb, experiments were performed in the presence of cold Rb plus 86Rb. Before the assay, tubules were preincubated for 10 min at 37 degrees C to restore the normal transmembrane cation gradients. 86Rb uptake was measured after washing out extracellular cations by rinsing the tubules in ice-cold choline chloride solution containing Ba2+. Rb uptake increased quasi-linearly as a function of incubation time up to 30 s in the thick ascending limb, 1 min in the proximal convoluted tubule, and 5 min in the collecting tubule, and reached an equilibrium after 5–;30 min. The initial rates of Rb uptake increased in a saturable fashion as Rb concentration in the medium rose from 0.25 to 5 mM. In medullary thick ascending limb, the initial rate of Rb uptake was inhibited by greater than 90% by 2.5 mM ouabain and by 10(-5) M of the metabolic inhibitor carbonyl cyanide trifluoromethoxyphenylhydrazone. Correlation of Na-K-ATPase hydrolytic activity at Vmax and initial rates of ouabain-sensitive Rb uptake in the successive segments of nephron indicates that in intact cells the pump works at approximately 20–30% of its Vmax. Increasing intracellular Na concentration by tubule preincubation in a Rb- and K-free medium increased the initial rates of Rb intake up to the Vmax of the hydrolytic activity of the pump.

Erythrocyte cation fluxes during the menstrual cycle in normal female subjects

1. Studies of erythrocyte cation transport mechanisms in vitro were performed on eight normotensive, premenopausal female subjects at the mid-points of the follicular and luteal phases of their menstrual cycles. Concurrent plasma concentrations of 17β-oestradiol, progesterone, aldosterone and renin activity were measured. 2. Ouabain-resistant, frusemide-resistant rubidium influx (an index of passive potassium diffusion) was significantly lower in the luteal than the follicular phase. 3. In further studies in four of the eight subjects, the mean rate constant of the rubidium influx measurement was also lower in the luteal than in the follicular phase. 4. There were no changes in Na+-K+ co-transport, sodium pump activity or intracellular cation concentrations throughout the cycle. 5. There was a tenfold fall in the mean plasma 17β-oestradiol/progesterone ratio, as well as increases in plasma aldosterone concentration and renin activity between the mid-follicular and mid-luteal phases. 6. We conclude that changes in plasma oestrogen/progesterone ratio during the menstrual cycle may be associated with alterations in passive potassium diffusion.

Response of the rat erythrocyte to ozone exposure

Sprague-Dawley rats were exposed to high (6--8 ppm) and moderate (1.5 ppm) amounts of ozone (O3) for various time periods. Response of the rat erythrocyte to ozone was monitored with red blood cell potassium (rubidium) influx studies, with storage stress combined with ultrastructural studies and with levels of erythrocyte glutathione peroxidase and superoxide dismutase. Erythrocytes of rats exposed to O3 showed no significant changes either in their potassium influx or in their glutathione peroxidase and superoxide dismutase activities compared to controls. Erythrocyte differential counts on O3-exposed animals showed significant changes initially as well as following storage stress compared to controls. Rats exposed to 8 ppm O3 for 4 h showed a marked increase in echinocytes. These consistent transformations from discocytes to echinocytes following O3 exposure suggest latent erythrocyte damage has occurred.

Response of the iron-deficient erythrocyte in the rat to hyperoxia

Normal and iron-deficient rats were exposed to 90% O2 at 760 Torr for 24 or 48 h. Erythrocyte response to hyperoxia was monitored by potassium (rubidium) influx studies, by storage stress, and by ultrastructural studies. Normal rat erythrocytes exhibited morphological changes and decrease of ouabain-sensitive potassium influx compared to unexposed controls. Both components of erythrocyte potassium influx were affected by iron deficiency. Erythrocytes from unexposed iron-deficient rats showed a 50% increase in ouabain-sensitive potassium influx compared to controls. Iron-deficient rats exposed to hyperoxia for 24 or 48 h, had erythrocytes with morphological changes. Erythrocytes of iron-deficient rats exposed for 24 h showed no influx change; those exposed for 48 h showed a decrease of ouabain-sensitive influx compared to erythrocytes of controls.