Douglas R. Yingst

Effect of hemolysate on calcium inhibition of the (Na+ + K+)-ATPase of human red blood cells

The sensitivity of the (Na+ + K+)-ATPase in human red cell membranes to inhibition by Ca2+ is markedly increased by the addition of diluted cytoplasm from hemolyzed human red blood cells. The concentration of Ca2+ causing 50% inhibition of the (Na+ + K+)-ATPase is shifted from greater than 50 microM free Ca2+ in the absence of hemolysate to less than 10 microM free Ca2+ when hemolysate diluted 1:60 compared to in vivo concentrations is added to the assay mixture. Boiling the hemolysate destroys its ability to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+. Proteins extracted from the membrane in the presence of EDTA and concentrated on an Amicon PM 30 membrane increased the sensitivity of the (Na+ + K+)-ATPase to Ca2+ in a dose-dependent fashion, causing over 80% inhibition of the (Na+ + K+)-ATPase at 10 microM free Ca2+ at the highest concentration of the extract tested. The active factor in this membrane extract is Ca2+-dependent, because it had no effect on the (Na+ + K+)-ATPase in the absence of Ca2+. Trypsin digestion prior to the assay destroyed the ability of this protein extract to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+.

Insights Into the Mechanism by Which Inhibition of Na,K-ATPase Stimulates Aldosterone Production

Inhibition of Na,K-adenosine triphosphatase (Na,K-ATPase) activity by ouabain has been shown to increase the release of aldosterone from rat glomerulosa cells, but the mechanism by which this elevation of aldosterone production occurs has not been established. Small changes in membrane potential can significantly affect aldosterone release. Consequently, inhibition of Na,K-ATPase in glomerulosa cells may stimulate aldosterone production by membrane depolarization. If so, ouabain-stimulated production should be dependent on calcium influx through voltage-gated calcium channels. It has previously been shown that ouabain induces a moderately rapid increase in cytosolic calcium in rat glomerulosa cells. Therefore, in this study, we test whether ouabain stimulates aldosterone production with a time course consistent with early membrane depolarization as suggested by the previously reported early increase in cytosolic calcium. To study the time course of aldosterone production, we developed a perfusion technique that allows an examination of the initial effects of ouabain on aldosterone production. The results show that ouabain rapidly stimulates aldosterone production. Continuous perfusion with 0.25 or 1 mmol/L ouabain induced a brisk, robust increase in aldosterone production, followed by a decrease to near baseline over 60 minutes. Ouabain-stimulated aldosterone production was dependent on the presence of extracellular calcium and calcium influx through voltage-gated calcium channels. Our results support the hypothesis that the inhibition of Na,K-ATPase in rat adrenal glomerulosa cells immediately depolarizes the membrane potential and opens voltage-gated calcium channels.

Purification of active Na+-K+-ATPase using a new ouabain-affinity column

Ouabain, a specific inhibitor of Na+-K+-ATPase, was coupled to epoxy agarose via a 13-atom spacer to make an affinity column that specifically binds Na+-K+-ATPase. Na+-K+-ATPase from rat and dog kidney was bound to the column and was eluted as a function of enzyme conformation, altered by adding specific combinations of ligands. Na+-K+-ATPase from both sources bound to the column in the presence of Na + ATP + Mg and in solutions containing 30 mM K. No binding was observed in the presence of Na or Na + ATP. These experiments suggest that Na+-K+-ATPase binds to the column under the same conditions that it binds to untethered ouabain. Na+-K+-ATPase already bound to the column was competitively eluted with excess free Na + ouabain or with Na + ATP. The latter eluted active enzyme. For comparable amounts of bound Na+-K+-ATPase, Na + ouabain and Na + ATP eluted more rat than dog Na+-K+-ATPase, consistent with the lower affinity of the rat Na+-K+-ATPase for ouabain. The ouabain-affinity column was used to purify active Na+-K+-ATPase from rat kidney microsomes and rat adrenal glomerulosa cells. The specific activity of the kidney enzyme was increased from ∼2 to 15 μmol Pi ⋅ mg-1 ⋅ min-1. Na+-K+-ATPase purified from glomerulosa cells that were prelabeled with [32P]orthophosphate was phosphorylated on the α-subunit, suggesting that these cells contain a kinase that phosphorylates Na+-K+-ATPase.

Inhibitors of tyrosine phosphatases block angiotensin II inhibition of Na+ pump

To determine how angiotensin II inhibits the Na(+) pump (Na(+), K(+)-ATPase) in rat zona glomerulosa, we selectively blocked signaling proteins that could be activated by the angiotensin AT(1) receptor and known to affect Na(+) pump activity. Inhibitors of protein kinase C [calphostin C (1 microM); staurosporine (1 microM)], phospholipase A(2) [arachidonyl triflouromethyl ketone (25 microM); quinacrine (75 microM)], diacylgycerol lipase [RHC-80267 (5 microM)], and tyrosine phosphorylation [tyrphostin 47 (100 microM)] had no effect on angiotensin II inhibition of the Na(+) pump. On the other hand, inhibitors of tyrosine phosphatases [phenylarsine oxide (5 microM) and 4-bromotetramisole oxalate (100 microM)] blocked angiotensin II inhibition, where as inhibitors of serine/threonine phosphatases [okadaic acid (1 microM) and microcystin (1.5 microM)] did not. Thus, angiotensin II inhibition of the Na(+) pump may in part be mediated by a tyrosine phosphatase.

The effect of ethanol on the passive Ca permeability of human red cell ghosts measured by means of arsenazo III

Ethanol in the range of 0.76-2.40 M caused an immediate increase in the Ca permeability of the plasma membrane of resealed human red blood cell ghosts in which intracellular free Ca could be continuously monitored by means of the Ca chromophore arsenazo III. At a given concentration of ethanol, the Ca permeability increased markedly a few minutes following the mixing of the ghosts and the ethanol, and continued to increase over at least the next 30 min. Preincubating the ghosts in ethanol for 15, 60 and 120 min before measuring the rate of free Ca accumulation, progressively increased the effect of a given concentration of ethanol. These results indicate that the effect of a given concentration of ethanol is a complex function of concentration and exposure time. The effects of ethanol in this concentration range were completely reversible. The resealed ghosts used in these experiments were depleted of ATP to avoid interference from the Ca pump and all experiments were carried out with 150 mM KCl on both sides of the membrane to minimize changes in either the volume or membrane potential associated with activation of the Ca-dependent K channel.

Hemolysate increases calcium-inhibition of the Na+,K+ pump of resealed human red cell ghosts

The Na+,K+ pump of resealed human red cell ghosts is more sensitive to inhibition by intracellular Ca (Cai) when they contain diluted hemolysate compared to ghosts without hemolysate. The activity of the Na+,K+ pump was assessed by measuring ouabain-sensitive 22Na efflux in ghosts that, in addition to the presence or absence of hemolysate, also contained arsenazo III to measure free Cai and a regenerating system to maintain a constant concentration of ATP. Incorporating hemolysate diluted 20-fold compared to in situ conditions doubled the inhibitory effects of 1-50 microM free Cai on the Na+,K+ pump and caused 50% inhibition to occur between 5 and 10 microM free Cai. Increased inhibition in the presence of the hemolysate was not due to a cytoplasm-induced decrease in the ATP content of the ghosts. These findings are consistent with the suggestion that the cytoplasm of human red cells contains a factor which increases the sensitivity of the Na+,K+ pump to inhibition by Cai.

Decreased renal perfusion rapidly increases plasma membrane Na-K-ATPase in rat cortex by an angiotensin II-dependent mechanism

To understand how rapid changes in blood pressure can regulate Na-K-ATPase in the kidney cortex, we tested the hypothesis that a short-term (5 min) decrease in renal perfusion pressure will increase the amount of Na-K-ATPase in the plasma membranes by an angiotensin II-dependent mechanism. The abdominal aorta of anesthetized Sprague-Dawley rats was constricted with a ligature between the renal arteries, and pressure was monitored on either side during acute constriction. Left renal perfusion pressure was reduced to 70 +/- 1 mmHg (n = 6), whereas right renal perfusion pressure was 112 +/- 4 mmHg. In control (nonconstricted) rats (n = 5), pressure to both kidneys was similar at 119 +/- 6 mmHg. After 5 min of reduced perfusion, femoral venous samples were taken for plasma renin activity (PRA) and the kidneys excised. The cortex was dissected, minced, sieved, and biotinylated. Lower perfusion left kidneys showed a 41% increase (P < 0.003) in the amount of Na-K-ATPase in the plasma membrane compared with right kidneys. In controls, there was no difference in cell surface Na-K-ATPase between left and right kidneys (P = 0.47). PRA was 57% higher in experimental animals compared with controls. To test the role of angiotensin II in mediating the increase in Na-K-ATPase, we repeated the experiments (n = 6) in rats treated with ramiprilat. When angiotensin-converting enzyme was inhibited, the cell surface Na-K-ATPase of the two kidneys was equal (P =0.46). These results confirm our hypothesis: rapid changes in blood pressure regulate trafficking of Na-K-ATPase in the kidney cortex.

Sensitivity and reversibility of Ca-dependent inhibition of the (Na++K+)-ATPase of human red blood cells

The sensitivity of the (Na+ + K+)-ATPase to inhibition by Ca was increased 30-fold by a partially purified extract of human red cell hemolysate. The hemolysate fraction reduced the concentration of free Ca required for 50% inhibition from 30 microM to approx. 1 microM. Ca-dependent inhibition of the (Na+ + K+)-ATPase in the presence and absence of the hemolysate fraction was completely reversible. The hemolysate fraction also stimulated the Ca2+-ATPase and increased its affinity for Ca. In the presence of the hemolysate fraction, the concentration of free Ca that inhibited the (Na+ + K+)-ATPase by 50% was similar to that which half-maximally stimulated the Ca2+-ATPase. Boiling the fraction destoryed its effect on the (Na+ + K+)-ATPase, but did not impair its stimulation of the Ca2+-ATPase.

Increased expression of Na,K-ATPase and a selective increase in phosphorylation at Ser-11 in the cortex of the 2-kidney, 1-clip hypertensive rat

BACKGROUND The mechanism by which blood pressure increases during renovascular hypertension is incompletely understood. We, therefore, tested the hypothesis that in the 2-kidney, 1-clip (2K-1C) rat, in which hypertension develops due to increased angiotensin II (Ang II) levels, there is increased expression and phosphorylation of Na,K-ATPase at Ser-11 and Ser-18 in the kidney cortex. The rationale is Ang II is reported to directly stimulate Na,K-ATPase activity in proximal tubules, which reabsorb 2/3 of filtered sodium, via increased phosphorylation at Ser-11 and Ser-18 and the Na,K-ATPase drives sodium reabsorption. METHODS Five-week-old Sprague-Dawley rats underwent unilateral or sham clipping of the right renal artery and placement of telemetry transmitters. Six weeks later blood pressure and plasma Ang II were measured and kidneys harvested. The amount of Na,K-ATPase, phosphorylation at Ser-11 and Ser-18, and the expression of β-actin in each kidney cortex were measured by quantitative immunoblotting. RESULTS Clipping significantly increased mean arterial pressure from 110 ± 3 to 148 ± 13 mm Hg, plasma Ang II, cortical Na,K-ATPase in the unclipped kidney of 2K-1C compared to sham-clipped rats, the total cortical Na,K-ATPase in both kidneys compared to sham-clipped rats, and the extent to which the Na,K-ATPase was phosphorylated at Ser-11. Clipping did not significantly change phosphorylation at Ser-18, β-actin, or the total protein in the cortexes of both kidneys. CONCLUSIONS Thus, in the kidney cortex of rats with renovascular hypertension there is increased expression of Na,K-ATPase and a selective increase in its phosphorylation at Ser-11 that could increase the capacity to reabsorb sodium and water.

Angiotensin II stimulates elution of Na-K-ATPase from a digoxin-affinity column by increasing the kinetic response to ligands that trigger the decay of E2-P

We earlier observed that treating rat proximal tubules with concentrations of angiotensin II (ANG II) that directly stimulate Na-K-ATPase activity changed how Na-K-ATPase subsequently eluted from an ouabain-affinity column. In this study we tested whether ANG II increases the rate of elution in response to ligands that trigger the decay of E(2)-P, which implies a change in functional properties of Na-K-ATPase, or by decreasing the amount subsequently eluted with SDS, which suggests a change in how Na-K-ATPase interacts with other proteins. We utilized a new digoxin-affinity column and novel lines of opossum kidney (OK) cells that coexpress the rat AT(1a) receptor and either the wild-type rat alpha(1)-isoform of Na-K-ATPase or a truncation mutant missing the first 32 amino acids of its NH(2) terminus. We characterized how rat kidney microsomes bind to and elute from the digoxin-affinity column and demonstrated that they are heterogeneous in the rate at which they release digoxin in response to ligands that trigger the decay of E(2)-P. Incubating OK cells with ANG II stimulated the ensuing elution of wild-type rat alpha(1)-subunit by increasing the kinetic response to ligands that cause a decay of E(2)-P without affecting the amount later eluted with SDS. In contrast, ANG II had no effect on the kinetic response of the truncation mutant but decreased the amount eluted with SDS. These data suggest that ANG II regulates both the kinetic properties of Na-K-ATPase and its interaction with other proteins by a mechanism(s) involving its NH(2) terminus.