Douglas Yingst - Academia.edu (original) (raw)

Papers by Douglas Yingst

The FASEB Journal, Mar 1, 2008

The FASEB Journal, Apr 1, 2007

American Journal of Physiology-renal Physiology, Nov 1, 2009

To understand how rapid changes in blood pressure can regulate Na-K-ATPase in the kidney cortex, ... more 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 Ϯ 1 mmHg (n ϭ 6), whereas right renal perfusion pressure was 112 Ϯ 4 mmHg. In control (nonconstricted) rats (n ϭ 5), pressure to both kidneys was similar at 119 Ϯ 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 Ͻ 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 ϭ 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 ϭ 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 ϭ0.46). These results confirm our hypothesis: rapid changes in blood pressure regulate trafficking of Na-K-ATPase in the kidney cortex. ouabain; sodium; blood pressure; renin THE PROXIMAL TUBULE REABSORBS approximately two-thirds of the filtered sodium, which contributes to the control of plasma blood volume and the regulation of blood pressure. The Na-K-ATPase is an active transport protein in the basolateral membrane that is widely recognized as playing a key role in sodium reabsorption. The hormone dopamine inhibits (4) and angiotensin II (ANG II) directly stimulates the short-term (Յ15 min) activity of the Na-K-ATPase (1, 5, 6, 7, 21), which eventually affects blood pressure. In opossum kidney cells, a cell culture model of proximal tubules, which express the ␣ 1-subunit of the rat kidney Na-K-ATPase, the short-term stimulation of Na-K-ATPase activity by ANG II is mediated by the AT 1 receptor, which stimulates PKC that in turn increases the phosphorylation of Na-K-ATPase and thereby triggers increased trafficking of Na-K-ATPase to the plasma membrane (5, 6).

Analytical Biochemistry, Jul 1, 1983

Arsenazo III in human red cell ghosts is calibrated to measure intracellular concentrations of fr... more Arsenazo III in human red cell ghosts is calibrated to measure intracellular concentrations of free Ca and free Mg. This calibration was established by comparing the absorbance of arsenazo III in ghosts to its absorption in solution at 600, 630, and 655 nm as a function of buffered free Ca (0.4 microM to 70 microM), free Mg (0.05 to 5 mM), and free Ca (4 to 50 microM) at constant free Mg (1.2 mM) at three concentrations of total dye (1.09, 10.9, and 109 microM). In both ghosts and in solution the absorbance of the dye at all three wavelengths could be predicted from dissociation constants and molar extinction coefficients determined for a 1:1 complex with the dye and Mg, another with Ca, and a third complex consisting of two molecules of Ca and two of dye. The absorbance of the dye in ghosts at the same concentrations of free Ca, free Mg, and total dye is equal to that in solution multiplied by the percentage hematocrit and divided by 100, which demonstrates that arsenazo III responds the same inside ghosts as it does free in solution. The results of this paper show that arsenazo III can be used to measure quantitatively and to monitor continuously the concentration of intracellular Ca and Mg in red cell ghosts. Use of this method should facilitate the study of Ca-dependent mechanisms of red blood cells.

American Journal of Physiology-cell Physiology, 2001

Effects of extracellular calcium and potassium on the sodium pump of rat adrenal glomerulosa cell... more Effects of extracellular calcium and potassium on the sodium pump of rat adrenal glomerulosa cells. Am J Physiol Cell Physiol 280: C119-C125, 2001.-Because the activity of the sodium pump (Na-K-ATPase) influences the secretion of aldosterone, we determined how extracellular potassium (K o) and calcium affect sodium pump activity in rat adrenal glomerulosa cells. Sodium pump activity was measured as ouabain-sensitive 86 Rb uptake in freshly dispersed cells containing 20 mM sodium as measured with sodium-binding benzofluran isophthalate. Increasing K o from 4 to 10 mM in the presence of 1.8 mM extracellular calcium (Ca o) stimulated sodium pump activity up to 165% and increased intracellular free calcium as measured with fura 2. Increasing K o from 4 to 10 mM in the absence of Ca o stimulated the sodium pump ϳ30% and did not increase intracellular free calcium concentration ([Ca 2ϩ ] i). In some experiments, addition of 1.8 mM Ca o in the presence of 4 mM K o increased [Ca 2ϩ ] i above the levels observed in the absence of Ca o and stimulated the sodium pump up to 100%. Cadependent stimulation of the sodium pump by K o and Ca o was inhibited by isradipine (10 M), a blocker of Land T-type calcium channels, by compound 48/80 (40 g/ml) and calmidizolium (10 M), which inhibits calmodulin (CaM), and by KN-62 (10 M), which blocks some forms of Ca/CaM kinase II (CaMKII). Staurosporine (1 M), which effectively blocks most forms of protein kinase C, had no effect. In the presence of A-23187, a calcium ionophore, the addition of 0.1 mM Ca o increased [Ca 2ϩ ] i to the level observed in the presence of 10 mM K o and 1.8 mM Ca o and stimulated the sodium pump 100%. Ca-dependent stimulation by A-23187 and 0.1 mM Ca o was not reduced by isradipine but was blocked by KN-62. Thus, under the conditions that K o stimulates aldosterone secretion, it stimulates the sodium pump by two mechanisms: direct binding to the pump and by increasing calcium influx, which is dependent on Ca o. The resulting increase in [Ca 2ϩ ] i may stimulate the sodium pump by activating CaM and/or CaMKII.

Annual Review of Physiology, Oct 1, 1988

The activity of the Na,K-ATPase can be sensitive to physiological changes in Cai. Intracellular p... more The activity of the Na,K-ATPase can be sensitive to physiological changes in Cai. Intracellular proteins such as calnaktin, calmodulin, and protein kinase C could regulate the pump during transient changes in Cai. The mechanisms by which these proteins interact with the Na,K-ATPase, their distribution in different kinds of cells, and their role in regulating the Na,K-ATPase are not yet determined. Preliminary data indicate that an increase in Cai within the physiological range could be associated with either a stimulation or an inhibition of enzyme activity or a change in the affinity of the Na,K-ATPase for ouabain. The type of response probably depends on the kind of cell, its associated intracellular proteins, and its physiological state. Ca and intracellular proteins could play a key role in the regulation of the Na,K-ATPase by hormones.

European Journal of Pharmacology, Oct 1, 2000

Ž q q. To determine how angiotensin II inhibits the Na pump Na , K-ATPase in rat zona glomerulosa... more Ž q q. 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 receptor and known to affect Na q pump activity. Inhibitors of protein kinase C q 2q

Biochimica Et Biophysica Acta - Biomembranes, Jul 1, 1983

The Na+,K + pump of resealed human red cell ghosts is more sensitive to inhibition by intracellul... more The Na+,K + pump of resealed human red cell ghosts is more sensitive to inhibition by intracellular Ca (Ca i) 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 Ca m 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 #M free Ca i on the Na+,K + pump and caused 50% inhibition to occur between 5 and 10/~M free Ca i. 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 Cal.

Biophysical Journal, Sep 1, 1978

Ca-sensitive dye, arsenazo III, has been incorporated into resealed human erythrocyte ghosts and ... more Ca-sensitive dye, arsenazo III, has been incorporated into resealed human erythrocyte ghosts and calibrated to monitor continuously micromolar concentrations of intracellular ionized Ca ([Ca+]). When the external concentration of Ca is much greater than [Ca'+ ]j, [Ca'+ ]i increases because of a net balance between Ca influx and efflux. Dynamic changes in [Ca +]i regulate K efflux, which in turn may influence the rate of Ca influx. A procedure for purifying arsenazo III is also described.

Biochimica Et Biophysica Acta - Biomembranes, Mar 1, 1985

Ethanol in the range of 0.76-2.40 M caused an immediate increase in the Ca permeability of the pl... more 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 rain. 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 KCI 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.

Metabolism-clinical and Experimental, Sep 1, 1999

Inhibition of Na,K-adenosine triphosphatase (Na,K-ATPase) activity by ouabain has been shown to i... more 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, ouabainstimulated 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 glomeruiosa cells immediately depolarizes the membrane potential and opens voltage-gated calcium channels.

The Biological Bulletin, Oct 1, 1974

Although holopelagic polychaetes are widely distributed throughout the oceans, very little quanti... more Although holopelagic polychaetes are widely distributed throughout the oceans, very little quantitative information exists on their vertical distribution and even less is known of their reproductive biology and life histories. Most of our knowl

American Journal of Physiology-renal Physiology, Apr 1, 2008

We earlier observed that treating rat proximal tubules with concentrations of angiotensin II (ANG... more 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 digoxinaffinity column and novel lines of opossum kidney (OK) cells that coexpress the rat AT 1a receptor and either the wild-type rat α 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 α 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. Keywords cardiac glycosides; ouabain; epithelial cells; sodium THE MECHANISMS BY WHICH angiotensin II (ANG II) directly stimulates the short-term activity of Na-K-ATPase in the proximal tubule (1,2,7) are incompletely understood, which limits our knowledge of how ANG II controls sodium reabsorption and how ANG II and angiotensinconverting enzyme (ACE) inhibitors affect the development of hypertension and the treatment of congestive heart failure (3,29). When cells are exposed to ANG II, at least part of the stimulation that occurs during the first 15 min is due to the accumulation of Na-K

American Journal of Hypertension, Apr 1, 2012

Background The mechanism by which blood pressure increases during renovascular hypertension is in... more 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.

The Journal of General Physiology, 1984

The rate of Ca influx into ghosts containing arsenazo III changes with time, being most rapid dur... more The rate of Ca influx into ghosts containing arsenazo III changes with time, being most rapid during the first 5 min after Ca is added to the outside and declining thereafter. The rate of Ca influx is a nonlinear function of extracellular Ca and plateaus as the latter is increased above 1 mM. The rate of Ca influx was measured as a function of the transmembrane gradients of Na and K and changes in the permeability of the membrane to K and Cl produced by valinomycin and SITS (4-acetamido-4'-isothiocyano-stilbene-2-2'-disulfonic acid), respectively. Changes in the rate of Ca influx are consistent with expected effects of these treatments on the membrane potential. Oligomycin (10 jug/ml) and quinidine (1 mM) inhibit the rate of Ca uptake by inhibiting Ca-induced changes in the K permeability. At constant membrane potential, furosemide produced a slight (15%) consistent increase in Ca uptake. Other experiments show that resealed ghosts are heterogeneous in their passive permeability to Ca and that A23187 can be used to effectively eliminate such differences. The results of this paper show that resealed human red cell ghosts containing arsenazo III can be used to continuously monitor intracellular free Ca and to study the factors that influence the permeability of the red cell membrane to Ca.

Biochimica Et Biophysica Acta - Biomembranes, Mar 1, 1985

The sensitivity of the (Na + + K +)-ATPase to inhibition by Ca was increased 30-fold by a partial... more 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 #M to approx. 1 #M. 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.

The Journal of General Physiology, 1984

Increasing free intracellular Ca (Ca;) from <0 .1 AM to 10 AM by means of A23187 activated Ca-sti... more Increasing free intracellular Ca (Ca;) from <0 .1 AM to 10 AM by means of A23187 activated Ca-stimulated K transport and inhibited the Na-K pump in resealed human red cell ghosts. These ghosts contained 2 mM ATP, which was maintained by a regenerating system, and arsenazo III to measure Ca ;. Ca-stimulated K transport was activated 50% at 2-3 FM free Ca; and the Na-K pump was inhibited 50% by 5-10 AM free Ca;. Free Ca ; from 1 to 8 AM stimulated K efflux before it inhibited the Na-K pump, dissociating the effect of Ca on the two systems. 3 AM trifluoperazine inhibited Ca-stimulated K efflux and 0.5 mM quinidine reduced Na-K pumping by 50%. In other studies, incubating fresh intact cells in solutions containing Ca and 0.5 AM A23187 caused the cells to lose K heterogeneously. Under the same conditions, increasing A23187 to 10 AM initiated a homogeneous loss of K. In ATP-deficient ghosts containing Ca; equilibrated with A23187, K transport was activated at the same free Ca ; as in the ghosts containing 2 mM ATP. Neither Ca. nor the presence of an inward Ca gradient altered the effect of free Ca; on the permeability to K. In these ghosts, transmembrane interactions of Na and K influenced the rate of Ca-stimulated K efflux independent of Na-and K-induced changes in free Cai or sensitivity to Ca;. At constant free Ca;, increasing Ko from 0.1 to 3 mM stimulated K efflux, whereas further increasing Ko inhibited it. Increasing Nai at constant Ki and free Ca; markedly decreased the rate of efflux at 2 mM K.o, but had no effect when K o was >_20 mM. These transmembrane interactions indicate that the mechanism underlying Ca-stimulated K transport is mediated. Since these interactions from either side of the membrane are independent of free Cai, activation of the transport mechanism by Ca; must be at a site that is independent of those responsible for the interaction of Na and K. In the presence of A23187, this activating site is half-maximally stimulated by^-2 AM free Ca and is not influenced by the concentration of ATP. The J. GEN .

American Journal of Physiology-cell Physiology, Feb 1, 2016

of rat kidney Na-K pump at Ser 938 is required for rapid angiotensin II-dependent stimulation of ... more of rat kidney Na-K pump at Ser 938 is required for rapid angiotensin II-dependent stimulation of activity and trafficking in proximal tubule cells.

Physiological Reports, Nov 1, 2022

The FASEB Journal, Apr 1, 2009

The FASEB Journal, Mar 1, 2008

The FASEB Journal, Apr 1, 2007

American Journal of Physiology-renal Physiology, Nov 1, 2009

To understand how rapid changes in blood pressure can regulate Na-K-ATPase in the kidney cortex, ... more 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 Ϯ 1 mmHg (n ϭ 6), whereas right renal perfusion pressure was 112 Ϯ 4 mmHg. In control (nonconstricted) rats (n ϭ 5), pressure to both kidneys was similar at 119 Ϯ 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 Ͻ 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 ϭ 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 ϭ 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 ϭ0.46). These results confirm our hypothesis: rapid changes in blood pressure regulate trafficking of Na-K-ATPase in the kidney cortex. ouabain; sodium; blood pressure; renin THE PROXIMAL TUBULE REABSORBS approximately two-thirds of the filtered sodium, which contributes to the control of plasma blood volume and the regulation of blood pressure. The Na-K-ATPase is an active transport protein in the basolateral membrane that is widely recognized as playing a key role in sodium reabsorption. The hormone dopamine inhibits (4) and angiotensin II (ANG II) directly stimulates the short-term (Յ15 min) activity of the Na-K-ATPase (1, 5, 6, 7, 21), which eventually affects blood pressure. In opossum kidney cells, a cell culture model of proximal tubules, which express the ␣ 1-subunit of the rat kidney Na-K-ATPase, the short-term stimulation of Na-K-ATPase activity by ANG II is mediated by the AT 1 receptor, which stimulates PKC that in turn increases the phosphorylation of Na-K-ATPase and thereby triggers increased trafficking of Na-K-ATPase to the plasma membrane (5, 6).

Analytical Biochemistry, Jul 1, 1983

Arsenazo III in human red cell ghosts is calibrated to measure intracellular concentrations of fr... more Arsenazo III in human red cell ghosts is calibrated to measure intracellular concentrations of free Ca and free Mg. This calibration was established by comparing the absorbance of arsenazo III in ghosts to its absorption in solution at 600, 630, and 655 nm as a function of buffered free Ca (0.4 microM to 70 microM), free Mg (0.05 to 5 mM), and free Ca (4 to 50 microM) at constant free Mg (1.2 mM) at three concentrations of total dye (1.09, 10.9, and 109 microM). In both ghosts and in solution the absorbance of the dye at all three wavelengths could be predicted from dissociation constants and molar extinction coefficients determined for a 1:1 complex with the dye and Mg, another with Ca, and a third complex consisting of two molecules of Ca and two of dye. The absorbance of the dye in ghosts at the same concentrations of free Ca, free Mg, and total dye is equal to that in solution multiplied by the percentage hematocrit and divided by 100, which demonstrates that arsenazo III responds the same inside ghosts as it does free in solution. The results of this paper show that arsenazo III can be used to measure quantitatively and to monitor continuously the concentration of intracellular Ca and Mg in red cell ghosts. Use of this method should facilitate the study of Ca-dependent mechanisms of red blood cells.

American Journal of Physiology-cell Physiology, 2001

Effects of extracellular calcium and potassium on the sodium pump of rat adrenal glomerulosa cell... more Effects of extracellular calcium and potassium on the sodium pump of rat adrenal glomerulosa cells. Am J Physiol Cell Physiol 280: C119-C125, 2001.-Because the activity of the sodium pump (Na-K-ATPase) influences the secretion of aldosterone, we determined how extracellular potassium (K o) and calcium affect sodium pump activity in rat adrenal glomerulosa cells. Sodium pump activity was measured as ouabain-sensitive 86 Rb uptake in freshly dispersed cells containing 20 mM sodium as measured with sodium-binding benzofluran isophthalate. Increasing K o from 4 to 10 mM in the presence of 1.8 mM extracellular calcium (Ca o) stimulated sodium pump activity up to 165% and increased intracellular free calcium as measured with fura 2. Increasing K o from 4 to 10 mM in the absence of Ca o stimulated the sodium pump ϳ30% and did not increase intracellular free calcium concentration ([Ca 2ϩ ] i). In some experiments, addition of 1.8 mM Ca o in the presence of 4 mM K o increased [Ca 2ϩ ] i above the levels observed in the absence of Ca o and stimulated the sodium pump up to 100%. Cadependent stimulation of the sodium pump by K o and Ca o was inhibited by isradipine (10 M), a blocker of Land T-type calcium channels, by compound 48/80 (40 g/ml) and calmidizolium (10 M), which inhibits calmodulin (CaM), and by KN-62 (10 M), which blocks some forms of Ca/CaM kinase II (CaMKII). Staurosporine (1 M), which effectively blocks most forms of protein kinase C, had no effect. In the presence of A-23187, a calcium ionophore, the addition of 0.1 mM Ca o increased [Ca 2ϩ ] i to the level observed in the presence of 10 mM K o and 1.8 mM Ca o and stimulated the sodium pump 100%. Ca-dependent stimulation by A-23187 and 0.1 mM Ca o was not reduced by isradipine but was blocked by KN-62. Thus, under the conditions that K o stimulates aldosterone secretion, it stimulates the sodium pump by two mechanisms: direct binding to the pump and by increasing calcium influx, which is dependent on Ca o. The resulting increase in [Ca 2ϩ ] i may stimulate the sodium pump by activating CaM and/or CaMKII.

Annual Review of Physiology, Oct 1, 1988

The activity of the Na,K-ATPase can be sensitive to physiological changes in Cai. Intracellular p... more The activity of the Na,K-ATPase can be sensitive to physiological changes in Cai. Intracellular proteins such as calnaktin, calmodulin, and protein kinase C could regulate the pump during transient changes in Cai. The mechanisms by which these proteins interact with the Na,K-ATPase, their distribution in different kinds of cells, and their role in regulating the Na,K-ATPase are not yet determined. Preliminary data indicate that an increase in Cai within the physiological range could be associated with either a stimulation or an inhibition of enzyme activity or a change in the affinity of the Na,K-ATPase for ouabain. The type of response probably depends on the kind of cell, its associated intracellular proteins, and its physiological state. Ca and intracellular proteins could play a key role in the regulation of the Na,K-ATPase by hormones.

European Journal of Pharmacology, Oct 1, 2000

Ž q q. To determine how angiotensin II inhibits the Na pump Na , K-ATPase in rat zona glomerulosa... more Ž q q. 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 receptor and known to affect Na q pump activity. Inhibitors of protein kinase C q 2q

Biochimica Et Biophysica Acta - Biomembranes, Jul 1, 1983

The Na+,K + pump of resealed human red cell ghosts is more sensitive to inhibition by intracellul... more The Na+,K + pump of resealed human red cell ghosts is more sensitive to inhibition by intracellular Ca (Ca i) 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 Ca m 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 #M free Ca i on the Na+,K + pump and caused 50% inhibition to occur between 5 and 10/~M free Ca i. 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 Cal.

Biophysical Journal, Sep 1, 1978

Ca-sensitive dye, arsenazo III, has been incorporated into resealed human erythrocyte ghosts and ... more Ca-sensitive dye, arsenazo III, has been incorporated into resealed human erythrocyte ghosts and calibrated to monitor continuously micromolar concentrations of intracellular ionized Ca ([Ca+]). When the external concentration of Ca is much greater than [Ca'+ ]j, [Ca'+ ]i increases because of a net balance between Ca influx and efflux. Dynamic changes in [Ca +]i regulate K efflux, which in turn may influence the rate of Ca influx. A procedure for purifying arsenazo III is also described.

Biochimica Et Biophysica Acta - Biomembranes, Mar 1, 1985

Ethanol in the range of 0.76-2.40 M caused an immediate increase in the Ca permeability of the pl... more 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 rain. 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 KCI 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.

Metabolism-clinical and Experimental, Sep 1, 1999

Inhibition of Na,K-adenosine triphosphatase (Na,K-ATPase) activity by ouabain has been shown to i... more 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, ouabainstimulated 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 glomeruiosa cells immediately depolarizes the membrane potential and opens voltage-gated calcium channels.

The Biological Bulletin, Oct 1, 1974

Although holopelagic polychaetes are widely distributed throughout the oceans, very little quanti... more Although holopelagic polychaetes are widely distributed throughout the oceans, very little quantitative information exists on their vertical distribution and even less is known of their reproductive biology and life histories. Most of our knowl

American Journal of Physiology-renal Physiology, Apr 1, 2008

We earlier observed that treating rat proximal tubules with concentrations of angiotensin II (ANG... more 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 digoxinaffinity column and novel lines of opossum kidney (OK) cells that coexpress the rat AT 1a receptor and either the wild-type rat α 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 α 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. Keywords cardiac glycosides; ouabain; epithelial cells; sodium THE MECHANISMS BY WHICH angiotensin II (ANG II) directly stimulates the short-term activity of Na-K-ATPase in the proximal tubule (1,2,7) are incompletely understood, which limits our knowledge of how ANG II controls sodium reabsorption and how ANG II and angiotensinconverting enzyme (ACE) inhibitors affect the development of hypertension and the treatment of congestive heart failure (3,29). When cells are exposed to ANG II, at least part of the stimulation that occurs during the first 15 min is due to the accumulation of Na-K

American Journal of Hypertension, Apr 1, 2012

Background The mechanism by which blood pressure increases during renovascular hypertension is in... more 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.

The Journal of General Physiology, 1984

The rate of Ca influx into ghosts containing arsenazo III changes with time, being most rapid dur... more The rate of Ca influx into ghosts containing arsenazo III changes with time, being most rapid during the first 5 min after Ca is added to the outside and declining thereafter. The rate of Ca influx is a nonlinear function of extracellular Ca and plateaus as the latter is increased above 1 mM. The rate of Ca influx was measured as a function of the transmembrane gradients of Na and K and changes in the permeability of the membrane to K and Cl produced by valinomycin and SITS (4-acetamido-4'-isothiocyano-stilbene-2-2'-disulfonic acid), respectively. Changes in the rate of Ca influx are consistent with expected effects of these treatments on the membrane potential. Oligomycin (10 jug/ml) and quinidine (1 mM) inhibit the rate of Ca uptake by inhibiting Ca-induced changes in the K permeability. At constant membrane potential, furosemide produced a slight (15%) consistent increase in Ca uptake. Other experiments show that resealed ghosts are heterogeneous in their passive permeability to Ca and that A23187 can be used to effectively eliminate such differences. The results of this paper show that resealed human red cell ghosts containing arsenazo III can be used to continuously monitor intracellular free Ca and to study the factors that influence the permeability of the red cell membrane to Ca.

Biochimica Et Biophysica Acta - Biomembranes, Mar 1, 1985

The sensitivity of the (Na + + K +)-ATPase to inhibition by Ca was increased 30-fold by a partial... more 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 #M to approx. 1 #M. 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.

The Journal of General Physiology, 1984

Increasing free intracellular Ca (Ca;) from <0 .1 AM to 10 AM by means of A23187 activated Ca-sti... more Increasing free intracellular Ca (Ca;) from <0 .1 AM to 10 AM by means of A23187 activated Ca-stimulated K transport and inhibited the Na-K pump in resealed human red cell ghosts. These ghosts contained 2 mM ATP, which was maintained by a regenerating system, and arsenazo III to measure Ca ;. Ca-stimulated K transport was activated 50% at 2-3 FM free Ca; and the Na-K pump was inhibited 50% by 5-10 AM free Ca;. Free Ca ; from 1 to 8 AM stimulated K efflux before it inhibited the Na-K pump, dissociating the effect of Ca on the two systems. 3 AM trifluoperazine inhibited Ca-stimulated K efflux and 0.5 mM quinidine reduced Na-K pumping by 50%. In other studies, incubating fresh intact cells in solutions containing Ca and 0.5 AM A23187 caused the cells to lose K heterogeneously. Under the same conditions, increasing A23187 to 10 AM initiated a homogeneous loss of K. In ATP-deficient ghosts containing Ca; equilibrated with A23187, K transport was activated at the same free Ca ; as in the ghosts containing 2 mM ATP. Neither Ca. nor the presence of an inward Ca gradient altered the effect of free Ca; on the permeability to K. In these ghosts, transmembrane interactions of Na and K influenced the rate of Ca-stimulated K efflux independent of Na-and K-induced changes in free Cai or sensitivity to Ca;. At constant free Ca;, increasing Ko from 0.1 to 3 mM stimulated K efflux, whereas further increasing Ko inhibited it. Increasing Nai at constant Ki and free Ca; markedly decreased the rate of efflux at 2 mM K.o, but had no effect when K o was >_20 mM. These transmembrane interactions indicate that the mechanism underlying Ca-stimulated K transport is mediated. Since these interactions from either side of the membrane are independent of free Cai, activation of the transport mechanism by Ca; must be at a site that is independent of those responsible for the interaction of Na and K. In the presence of A23187, this activating site is half-maximally stimulated by^-2 AM free Ca and is not influenced by the concentration of ATP. The J. GEN .

American Journal of Physiology-cell Physiology, Feb 1, 2016

of rat kidney Na-K pump at Ser 938 is required for rapid angiotensin II-dependent stimulation of ... more of rat kidney Na-K pump at Ser 938 is required for rapid angiotensin II-dependent stimulation of activity and trafficking in proximal tubule cells.

Physiological Reports, Nov 1, 2022

The FASEB Journal, Apr 1, 2009