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1.
Physiol Rep ; 10(21): e15508, 2022 11.
Article in English | MEDLINE | ID: mdl-36377055

ABSTRACT

Angiotensin II (Ang II)-dependent stimulation of the AT1 receptor in proximal tubules increases sodium reabsorption and blood pressure. Reabsorption is driven by the Na,K-pump that is acutely stimulated by Ang II, which requires phosphorylation of serine-938 (S938). This site is present in humans and only known to phosphorylated by PKA. Yet, activation of AT1 decreases cAMP required to activate PKA and inhibiting PKA does not block Ang II-dependent phosphorylation of S938. We tested the hypothesis that Ang II-dependent activation is mediated via increased phosphorylation at S938 through a PI3K/AKT-dependent pathway. Experiments were conducted using opossum kidney cells, a proximal tubule cell line, stably co-expressing the AT1 receptor and either the wild-type (α-1.wild-type) or an alanine substituted (α-1.S938A) form of rat kidney Na,K-pump. A 5-min exposure to 10 pM Ang II significantly activated Na,K-pump activity (56%) measured as short-circuit current across polarized α-1.wild-type cells. Wortmannin, at a concentration that selectively inhibits PI3K, blocked that Ang II-dependent activation. Ang II did not stimulate Na,K-pump activity in α-1.S938A cells. Ang II at 10 and 100 pM increased phosphorylation at S938 in α-1.wild-type cells measured in whole cell lysates. The increase was inhibited by wortmannin plus H-89, an inhibitor of PKA, not by either alone. Ang II activated AKT inhibited by wortmannin, not H-89. These data support our hypothesis and show that Ang II-dependent phosphorylation at S938 stimulates Na,K-pump activity and transcellular sodium transport.


Subject(s)
Angiotensin II , Phosphatidylinositol 3-Kinases , Rats , Animals , Humans , Angiotensin II/pharmacology , Angiotensin II/metabolism , Phosphorylation , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Serine/metabolism , Wortmannin/pharmacology , Wortmannin/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Kidney Tubules, Proximal/metabolism , Sodium/metabolism , Opossums/metabolism
2.
Am J Physiol Cell Physiol ; 310(3): C227-32, 2016 Feb 01.
Article in English | MEDLINE | ID: mdl-26582472

ABSTRACT

How angiotensin (ANG) II acutely stimulates the Na-K pump in proximal tubules is only partially understood, limiting insight into how ANG II increases blood pressure. First, we tested whether ANG II increases the number of pumps in plasma membranes of native rat proximal tubules under conditions of rapid activation. We found that exposure to 100 pM ANG II for 2 min, which was previously shown to increase affinity of the Na-K pump for Na and stimulate activity threefold, increased the amount of the Na-K pump in plasma membranes of native tubules by 33%. Second, we tested whether previously observed increases in phosphorylation of the Na-K pump at Ser(938) were part of the stimulatory mechanism. These experiments were carried out in opossum kidney cells, cultured proximal tubules stably coexpressing the ANG type 1 (AT1) receptor, and either wild-type or a S938A mutant of rat kidney Na-K pump under conditions found by others to stimulate activity. We found that 10 min of incubation in 10 pM ANG II stimulated activity of wild-type pumps from 2.3 to 3.5 nmol K · mg protein(-1) · min(-1) and increased the amount of the pump in the plasma membrane by 80% but had no effect on cells expressing the S938A mutant. We conclude that acute stimulation of Na-K pump activity in native rat proximal tubules includes increased trafficking to the plasma membrane and that phosphorylation at Ser(938) is part of the mechanism by which ANG II directly stimulates activity and trafficking of the rat kidney Na-K pump in opossum kidney cells.


Subject(s)
Angiotensin II/pharmacology , Cell Membrane/drug effects , Kidney Tubules, Proximal/drug effects , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Cell Line , Cell Membrane/enzymology , Cyclic AMP-Dependent Protein Kinases/metabolism , Enzyme Activation , Kidney Tubules, Proximal/enzymology , Male , Mutation , Opossums , Phosphorylation , Protein Kinase C/metabolism , Protein Transport , Rats, Sprague-Dawley , Receptor, Angiotensin, Type 1/agonists , Receptor, Angiotensin, Type 1/genetics , Receptor, Angiotensin, Type 1/metabolism , Serine , Sodium-Potassium-Exchanging ATPase/genetics , Time Factors , Transfection , Up-Regulation
3.
Am J Hypertens ; 25(4): 487-91, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22237155

ABSTRACT

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 ß-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, ß-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.


Subject(s)
Angiotensin II/blood , Hypertension, Renovascular/physiopathology , Kidney Cortex/metabolism , Sodium-Potassium-Exchanging ATPase/biosynthesis , Actins , Amino Acid Sequence , Angiotensin II/metabolism , Animals , Blood Pressure/drug effects , Constriction, Pathologic , Kidney Tubules, Proximal/metabolism , Male , Phosphorylation , Rats , Rats, Sprague-Dawley , Renal Artery/pathology , Serine/metabolism
4.
Biochem J ; 443(1): 249-58, 2012 Apr 01.
Article in English | MEDLINE | ID: mdl-22145807

ABSTRACT

Kidney plasma membranes, which contain a single α-1 isoform of Na+/K+-ATPase, simultaneously contain two sub-conformations of E2P, differing in their rate of digoxin release in response to Na+ and ATP. Treating cells with Ang II (angiotensin II) somehow changes the conformation of both, because it differentially inhibits the rate of digoxin release. In the present study we tested whether Ang II regulates release by increasing phosphorylation at Ser11/Ser18 and Ser938. Opossum kidney cells co-expressing the AT1a receptor and either α-1.wild-type, α-1.S11A/S18A or α-1.S938A were treated with or without 10 nM Ang II for 5 min, increasing phosphorylation at the three sites. Na+/K+-ATPase was bound to digoxin-affinity columns in the presence of Na+, ATP and Mg2+. A solution containing 30 mM NaCl and 3 mM ATP eluted ~20% of bound untreated Na+/K+-ATPase (Population #1). Pre-treating cells with Ang II slowed the elution of Population #1 in α-1.wild-type and α-1.S938A, but not α-1.S11A/S18A cells. Another 50% of bound Na+/K+-ATPase (Population #2) was subsequently eluted in two phases by a solution containing 150 mM NaCl and 3 mM ATP. Ang II increased the initial rate and slowed the second phase in α-1.wild-type, but not α-1.S938A, cells. Thus Ang II changes the conformation of two forms of EP2 via differential phosphorylation.


Subject(s)
Angiotensin II/chemistry , Serine/chemistry , Sodium-Potassium-Exchanging ATPase/isolation & purification , Amino Acid Substitution , Angiotensin II/pharmacology , Angiotensin II/physiology , Animals , Antibodies/chemistry , Blotting, Western , Cell Line , Chromatography, Affinity , Digoxin/chemistry , Kidney/cytology , Kidney/enzymology , Kinetics , Mutagenesis, Site-Directed , Opossums , Phosphorylation , Protein Binding , Protein Conformation , Rabbits , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium-Potassium-Exchanging ATPase/genetics , Sodium-Potassium-Exchanging ATPase/metabolism
5.
Am J Physiol Renal Physiol ; 297(5): F1324-9, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19726543

ABSTRACT

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.


Subject(s)
Angiotensin II/physiology , Cell Membrane/enzymology , Kidney Cortex/blood supply , Kidney Cortex/enzymology , Renal Circulation/physiology , Sodium-Potassium-Exchanging ATPase/metabolism , Angiotensin II/biosynthesis , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Animals , Biotin/metabolism , Blood Pressure/physiology , In Vitro Techniques , Male , Microsomes/enzymology , Ramipril/analogs & derivatives , Ramipril/pharmacology , Rats , Rats, Sprague-Dawley , Regional Blood Flow/drug effects , Renin/blood , Renin/physiology
6.
Am J Physiol Renal Physiol ; 294(4): F990-F1000, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18272598

ABSTRACT

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.


Subject(s)
Angiotensin II/pharmacology , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Chromatography, Affinity , Digoxin , Dinoprostone/pharmacology , Kidney/enzymology , Kidney Tubules, Proximal/enzymology , Kinetics , Microsomes/enzymology , Rats , Sodium-Potassium-Exchanging ATPase/isolation & purification
7.
Am J Physiol Renal Physiol ; 287(4): F713-21, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15161602

ABSTRACT

We present evidence that Na-K-ATPase in the rat proximal tubule is directly activated by ANG II much faster than previously observed. Specifically, we show that a 2-min exposure to 0.1 and 1 nM ANG II slowed the rate of intracellular sodium accumulation in response to an increase in extracellular sodium added in the presence of gramicidin D. From these data, we show that ANG II directly stimulates Na-K-ATPase activity at rate-limiting concentrations of intracellular sodium. Under these same conditions, exposing proximal tubules to ANG II altered the amount of 32P incorporated into multiple phosphopeptides generated from a tryptic digest of the alpha-subunit of Na-K-ATPase. Na-K-ATPase was isolated from whole cell lysates by means of a ouabain-affinity column and then separated into its individual subunits by SDS-PAGE. Na-K-ATPase bound to the column in its E2 conformation and was eluted by altering its conformation to E1 using Na+ATP. Na-K-ATPase isolated from cells treated with ANG II eluted more easily from the ouabain-affinity column than Na-K-ATPase isolated from control cells, suggesting that ANG II decreased the affinity of Na-K-ATPase for ouabain. Thus ANG II rapidly stimulated the activity of Na-K-ATPase in 2 min or less by a mechanism that could involve changes in phosphorylation and conformation of Na-K-ATPase. We suggest that the physiological role for rapid direct activation of Na-K-ATPase is greater control of intracellular sodium during sodium reabsorption.


Subject(s)
Angiotensin II/pharmacology , Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/enzymology , Sodium-Potassium-Exchanging ATPase/metabolism , Vasoconstrictor Agents/pharmacology , Animals , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Hypertension, Renal/metabolism , Male , Ouabain/metabolism , Ouabain/pharmacology , Phosphorylation/drug effects , Rats , Rats, Sprague-Dawley , Sodium/metabolism
8.
Proc Natl Acad Sci U S A ; 99(22): 14572-7, 2002 Oct 29.
Article in English | MEDLINE | ID: mdl-12388775

ABSTRACT

This study is aimed at identifying the Na pump isoform composition of human erythroid precursor cells and mature human erythrocytes. We used purified and synchronously growing human erythroid progenitor cells cultured for 7-14 days. RNA was extracted from the progenitor cells on different days and analyzed by RT-PCR. The results showed that only the alpha1, alpha3, beta2, and beta3 subunit isoforms and the gamma modulator were present. Northern analysis of the erythroid progenitor cells again showed that beta2 but not beta1 or alpha2 isoforms were present. The erythroid cells display a unique beta subunit expression profile (called beta-profiling) in that they contain the message for the beta2 isoform but not beta1, whereas leukocytes and platelets are known to have the message for the beta1 but not for the beta2 isoform. This finding is taken to indicate that our preparations are essentially purely erythroid and free from white cell contamination. Western analysis of these cultured progenitor cells confirmed the presence of alpha1, alpha3, (no alpha2), beta2, beta3, and gamma together now with clear evidence that beta1 protein was also present at all stages. Western analysis of the Na pump from mature human erythrocyte ghosts, purified by ouabain column chromatography, has also shown that alpha1, alpha3, beta1, beta2, beta3, and gamma are present. Thus, the Na pump isoform composition of human erythroid precursor cells and mature erythrocytes contains the alpha1 and alpha3 isoforms of the alpha subunit, the beta1, beta2, and beta3 isoforms of the beta subunit, and the gamma modulator.


Subject(s)
Erythrocytes/enzymology , Erythroid Precursor Cells/enzymology , Sodium-Potassium-Exchanging ATPase/metabolism , Cells, Cultured , Erythrocytes/cytology , Erythroid Precursor Cells/cytology , Humans , Isoenzymes , RNA, Messenger , Sodium-Potassium-Exchanging ATPase/genetics
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