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(Circulation Research. 2004;95:1100.)
© 2004 American Heart Association, Inc.

Cellular Biology

Hypertension-Linked Mutation in the Adducin -Subunit Leads to Higher AP2-µ2 Phosphorylation and Impaired Na⁺,K⁺-ATPase Trafficking in Response to GPCR Signals and Intracellular Sodium

Riad Efendiev, Rafael T. Krmar, Goichi Ogimoto, Jean Zwiller, Grazia Tripodi, Adrian I. Katz, Giuseppe Bianchi, Carlos H. Pedemonte, Alejandro M. Bertorello

From the Department of Medicine (R.T.K., G.O., A.M.B), Atherosclerosis Research Unit, Membrane Signaling Networks, Karolinska Institutet, Karolinska University Hospital, S-171 76 Stockholm, Sweden; the Department of Pharmacological and Pharmaceutical Sciences (R.E., C.H.P), College of Pharmacy, University of Houston, Tex; INSERM Unité 575 (J.Z.), Strasbourg, France; Prassis Research Institute Sigma-Tau (G.T.), Settimo Milanese, Italy; the Department of Medicine (A.I.K), University of Chicago, Ill; and Chair of Nephrology (G.B.), San Raffaele Hospital "Vita e Salute" University, Milan, Italy.

Correspondence to Dr Alejandro M. Bertorello, Department of Medicine, Atherosclerosis Research Unit, Karolinska University Hospital M1, S-171 76 Stockholm, Sweden. E-mail alejandro.bertorello{at}medks.ki.se

	Abstract

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-Adducin polymorphism in humans is associated with abnormal renal sodium handling and high blood pressure. The mechanisms by which mutations in adducin affect the renal set point for sodium excretion are not known. Decreases in Na⁺,K⁺-ATPase activity attributable to endocytosis of active units in renal tubule cells by dopamine regulates sodium excretion during high-salt diet. Milan rats carrying the hypertensive adducin phenotype have a higher renal tubule Na⁺,K⁺-ATPase activity, and their Na⁺,K⁺-ATPase molecules do not undergo endocytosis in response to dopamine as do those of the normotensive strain. Dopamine fails to promote the interaction between adaptins and the Na⁺,K⁺-ATPase because of adaptin-µ2 subunit hyperphosphorylation. Expression of the hypertensive rat or human variant of adducin into normal renal epithelial cells recreates the hypertensive phenotype with higher Na⁺,K⁺-ATPase activity, µ2-subunit hyperphosphorylation, and impaired Na⁺,K⁺-ATPase endocytosis. Thus, increased renal Na⁺,K⁺-ATPase activity and altered sodium reabsorption in certain forms of hypertension could be attributed to a mutant form of adducin that impairs the dynamic regulation of renal Na⁺,K⁺-ATPase endocytosis in response to natriuretic signals.

Key Words: high blood pressure • sodium retention • protein trafficking • cytoskeleton • endocytosis

	Introduction

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Spontaneous mutations in -adducin gene favor renal sodium retention and the development of high blood pressure in humans and rodents.¹ The estimated frequency of the mutated allele fluctuates among 8% in African Americans, 15% to 20% in whites, and 50% in Japanese individuals. The resulting phenotype is similar in rats and humans despite species differences in the mutation sites (human G460W/S586C; rats F316Y). These polymorphisms affect the pressure natriuresis curve, the lithium clearance (an indicator of proximal tubule [PCT] sodium transport),² and the chronic blood pressure response to diuretics.^3,4 Recent studies further substantiate the role of adducin in human hypertension and the associated renal or cardiac alterations.^5,6 Many of these findings have been obtained in low-renin hypertensive patients,⁷ in whom renal Na⁺ retention is a particularly important pathogenetic factor, or in patients carrying the gene variants known to affect renal Na⁺ handling.⁸

At the cellular level, adducin plays an active role in cytoskeleton organization by regulating actin dynamics.⁹ The mutated hypertensive variant of the adducin -subunit increases the rate of actin polymerization and bundling in a cell-free system compared with the wild-type variant.¹⁰ In cultured renal cells stably transfected with the mutated -adducin, the actin cytoskeleton network appears thicker and Na⁺,K⁺-ATPase activity is higher than in cells transfected with the wild-type variant.¹⁰

In renal epithelial cells, the Na⁺,K⁺-ATPase is located at the basolateral membrane domain and is critical for vectorial sodium transport. Its regulation by catecholamines contributes to the mechanisms that modulate urinary sodium excretion during salt loading or deprivation.¹¹ The Na⁺,K⁺-ATPase activity along the nephron is regulated in short- or long-term fashion by several hormones.^12,13 At the cellular level, increased renal tubule Na⁺,K⁺-ATPase activity^14,15 as well as an increased number of Na⁺,K⁺-ATPase units in the plasma membrane of cells of renal origin¹⁰ have been associated with functional abnormalities leading to high blood pressure. In addition, failure of natriuretic hormones to decrease Na⁺,K⁺-ATPase activity from renal PCT cells has been linked to the renal abnormalities leading to high blood pressure in Dahl salt-sensitive rats.^16,17 Because dopamine (DA)-dependent inhibition of Na⁺,K⁺-ATPase activity is mediated by internalization of its subunits^18–20 and this effect requires a permissive actin-microtubule cytoskeleton,¹⁸ we hypothesized that deficient endocytosis, by affecting the time that Na⁺,K⁺-ATPase molecules reside in the plasma membrane (and thereby vectorial sodium transport and reabsorption), may constitute the underlying mechanism for the abnormalities in renal PCT sodium transport seen with the development of high blood pressure. Identification of the tubular cell mechanisms responsible for controlling sodium reabsorption that are affected by -adducin polymorphism is important in helping to dissect some of the genetic mechanisms responsible for human hypertension.

	Materials and Methods

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Isolation of PCT Cells
PCT cells were isolated from 40- to 45-day-old Milan normotensive strain (MNS) and Milan hypertensive strain (MHS) rats weighing 150 to 250 g as described previously.¹⁸ Rats were bred at the Prassis-Research Institute Sigma-Tau animal facility and maintained at the Karolinska Institutet in the same conditions as described by Barber et al.²¹

Plasmids
An expression vector for hemagglutinin (HA)-tagged human -adducin (aa1-737, pCMVneoHA–ADD) was kindly provided by Dr Van Bennett (Duke University Medical Center, Durham, NC). Site-directed mutagenesis was performed on this construct to obtain the human hypertensive and normotensive variants of -adducin. The mutants were generated by exchanging nucleotides as follows: G460W (GGG versus TGG) and S586C (TGT versus TCT). The HA-tagged -adducin from MHS (Y316) and MNS (F316) were obtained by cDNA amplification and subcloning into the NheI–XhoI sites of the pCMVneoHA vector. Mutations were confirmed by DNA sequencing.

Cell Culture and Transfection
Opossum kidney (OK) cells stably transfected with rat Na⁺,K⁺-ATPase -subunit were cultured in 100-mm tissue culture dishes (80% confluent) containing 5 mL DMEM/10% bovine serum. Transient transfection with the different adducin cDNAs was performed using lipofection.²² The different forms of adducin carried a tag (HA), and an antibody against this tag was used to monitor expression levels during transient transfection.

Isolation of Endosomes and Basolateral Membranes
Isolated PCT cells from MNS or MHS rats and OK cells in suspension (1.5 mg protein/mL) were incubated for 15 minutes at room temperature with DA or vehicle (Hanks’ solution). Endosomes were fractionated on a flotation gradient,¹⁸ and basolateral membranes were prepared from a fraction obtained at the 16% and 42% sucrose interface of the flotation gradient using a Percoll gradient as described previously.¹⁸

Determination of Na⁺,K⁺-ATPase Activity
Isolated PCT cells were incubated with DA or vehicle (Hanks’ medium), and Na⁺,K⁺-ATPase hydrolytic activity was determined as the rate of ³²P-ATP hydrolysis in aliquots (1 to 3 µg protein).¹⁸ Na⁺,K⁺-ATPase activity was also determined in intact cells as the rate of rubidium transport.²³

Determination of Protein Phosphatase Activity
Nontransfected OK cells, and those transfected with the adducin wild-type or hypertensive variant cDNAs, were homogenized, centrifuged at 100 000g (4°C during 20 minutes), and protein phosphatase (PPase) activity toward phospho-histone was determined in the supernatant, as described.^22,24

Miscellaneous
All incubations with 1 µmol/L DA (diluted in Hanks’ vehicle) were performed at room temperature. Phosphorylation of Na⁺,K⁺-ATPase -subunit and of AP2 (adaptor protein 2)-µ2 subunit is described in detail in the online data supplement, available online at http://circres.ahajournals.org. Protein biotin labeling to separate the plasma membrane pool of Na⁺,K⁺-ATPase²³ and immunoprecipitation of AP2²⁵ (polyclonal antibody cat 06-552; Upstate Biotechnology) was performed as described. Immunoprecipitation of the p85 subunit of phosphatidylinositol 3-kinase (PI3K) was performed as described²⁶ using an antibody (cat sc-423) from Santa Cruz Biotechnology. Protein content was determined according to Bradford.²⁷ Western blots were developed with an ECL Plus kit (Amersham Pharmacia Biotech). The Na⁺,K⁺-ATPase -subunit was always identified in Western blots with a specific antibody provided by M.J. Caplan (Yale University, New Haven, Conn). Quantitation of Western blots was performed as described previously.¹⁸ Confocal microscopy²⁷ is described in the online data supplement.

Statistical Analysis
Statistical analysis of the data were performed with the unpaired Student t test. P values <0.05 were considered significant. In all figures, bars represent mean+SE.

	Results

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Na⁺,K⁺-ATPase Activity and Endocytosis in Renal Cells From MNS and MHS Rats
Na⁺,K⁺-ATPase activity was higher in PCT cells isolated from MHS than from MNS rats (Figure 1a, left), consistent with our previous observations.¹⁴ When PCT cells were incubated with DA, a significant inhibition of enzyme activity occurred in cells from MNS (Figure 1a, right). The degree of inhibition was similar to previous observations and within the expected range for a physiological response. In contrast, the inhibitory effect of DA was absent in cells derived from MHS rats (Figure 1a, right). Changes in Na⁺,K⁺-ATPase activity induced by membrane receptors or second messengers result from changes in the distribution of active molecules between the plasma membrane and intracellular compartments.^{18–20,23,25} Because the DA-induced decrease in Na⁺,K⁺-ATPase activity is caused by removal of active units from the basolateral plasma membrane (BLM),¹⁸ we examined whether failure of DA to reduce Na⁺,K⁺-ATPase activity in MHS cells was associated with impaired Na⁺,K⁺-ATPase endocytosis. DA reduced Na⁺,K⁺-ATPase -subunit within the BLM of PCT cells from MNS but not from MHS rats (Figure 1b). The decrease in BLM Na⁺,K⁺-ATPase -subunit was accompanied by an increased abundance in endosomes from MNS, whereas no change was observed in endosomes from MHS (Figure 1c). The and ß subunits of the Na⁺,K⁺-ATPase behave in parallel fashion during endocytosis.¹⁸ For simplicity, in this study, the -subunit was chosen as indicative of Na⁺,K⁺-ATPase endocytosis.

View larger version (48K): [in this window] [in a new window]   Figure 1. Effect of DA on Na+,K+-ATPase activity and endocytosis in PCT cells from MNS and MHS rats. a, Baseline enzyme activity in isolated PCT cells (left). Isolated PCT cells were incubated in the presence of DA or vehicle (right). Na+,K+-ATPase hydrolytic activity (nmol phosphate/mg protein per minute) is expressed as percentage of vehicle-treated PCT cells. *P<0.05; n=4. b and c, Isolated PCT cells were incubated with DA or vehicle, and Na+,K+-ATPase -subunit immunoreactivity was examined in BLM (b) and late endosomes (c). Representative Western blots (left panels) and their quantitation (right panels) are shown (n=5; five animals). *P<0.05. Na+,K+-ATPase -subunit phosphorylation (d), interaction of Na+,K+-ATPase with PI3K (e), and binding of AP2 to the Na+,K+-ATPase -subunit (f) in isolated cells incubated with DA or vehicle for 2 minutes. Representative blots are shown in the left panels and their quantitation in the right panels. *P<0.05; n=3.

Impaired Na⁺,K⁺-ATPase -Subunit–AP2 Interaction in MHS
To determine whether this abnormal response to DA in MHS was attributable to a defect in the DA receptor molecule or in its signaling responses, we examined several parameters: (1) DA increases Na⁺,K⁺-ATPase -subunit phosphorylation in DA-incubated PCT cells,¹⁹ and this increase is similar in cells from MNS or MHS (Figure 1d); (2) DA-dependent endocytosis of Na⁺,K⁺-ATPase requires activation of PI3K²⁰ through its interaction with a proline-rich motif within the Na⁺,K⁺-ATPase -subunit.²⁶ Here, we observed that DA promoted the interaction of PI3K p85 subunit with the Na⁺,K⁺-ATPase -subunit similarly in PCT cells isolated from MNS and MHS (Figure 1e); and (3) Products of PI3K such as phosphatidylinositol 3–phosphate mediate clathrin-dependent endocytosis of Na⁺,K⁺-ATPase, by promoting the association of adaptins (AP2) with a Tyr motif (Tyr-537), localized within the Na⁺,K⁺-ATPase -subunit.^25,28 Whereas in MNS, DA promoted the interaction of AP2 with the Na⁺,K⁺-ATPase -subunit, this effect was absent in cells from MHS (Figure 1f). These observations suggest that the lack of Na⁺,K⁺-ATPase endocytosis might be attributable to a defect beyond the DA receptor signal, at least one not involving phosphorylation of the -subunit, nor the initial steps of endocytosis, such as activation of PI3K. However, it seems to affect the later stages of endocytosis such as binding of AP2 and formation of the clathrin vesicles.

Hypertensive -Adducin Impairs Na⁺,K⁺-ATPase Endocytosis in a Normal Renal Cell Line
To determine whether the lack of changes in Na⁺,K⁺-ATPase activity and endocytosis in response to DA in MHS rats is directly associated with the mutation in the adducin molecule (and independent of the Na⁺,K⁺-ATPase or other abnormalities within the PCT cells from MHS), we expressed transiently in OK cells either form of rat adducin (-adducin wild-type [-WT]; hypertensive F316Y). Na⁺,K⁺-ATPase activity from vehicle-treated OK cells was similar in all groups except in cells expressing the F316Y, in which it was significantly higher (Figure 2a, left). DA inhibited Na⁺,K⁺-ATPase activity to the same extent (40%) in nontransfected, mock-transfected, and -WT but not in F316Y-transfected (15%) OK cells (Figure 2a, right). DA treatment led to a decrease in Na⁺,K⁺-ATPase -subunit abundance in the BLM of -WT but not of F316Y-transfected cells (Figure 2b). This was accompanied by an increase in Na⁺,K⁺-ATPase -subunit abundance only in endosomes of OK cells expressing the -WT. Similar to Milan rats, DA increased the Na⁺,K⁺-ATPase -subunit in the material immunoprecipitated with an AP2 antibody in nontransfected OK cells, mock-transfected OK cells, and in OK cells expressing the -WT but not the F316Y (Figure 2c). These studies further suggest that the presence of the hypertensive adducin prevents the interaction of AP2 with the Na⁺,K⁺-ATPase -subunit. Similar to MHS, the studies in cultured cells confirm that the AP2–Na⁺,K⁺-ATPase -subunit interaction is impaired in response to DA because of the presence of mutated adducin molecule.

View larger version (35K): [in this window] [in a new window]   Figure 2. Effect of DA on Na+,K+-ATPase activity and -subunit endocytosis in OK cells transiently expressing adducin -WT or hypertensive F316Y form. Nontransfected, mock-transfected, -WT, and F316Y-transfected OK cells were used. a, Na+,K+-ATPase activity was determined as the rate of 86Rb+ transport (nmol rubidium/mg protein per minute) in vehicle- and DA-treated cells for 5 minutes (n=3; performed in triplicate). Left panel shows the Na+,K+-ATPase activity from vehicle-treated cells, and the right panel indicates the percentage of inhibition in the presence of DA (*P<0.01). b, Na+,K+-ATPase -subunit abundance in BLM (left panel) and endosomes (right panel) from OK cells stably transfected with the -WT or F316Y. OK cells were incubated with vehicle or DA for 15 minutes (to allow the Na+,K+-ATPase molecules to reach the late endosomal compartment); n=4; *P<0.05. Because of the requirement of large quantities of transfected cells, the experiments in plasma membrane and endosomes were performed in cells expressing stably either adducin subunit. c, Nontransfected, mock-transfected, -WT, and F316Y-transfected OK cells were incubated with DA for 2.5 minutes. The material immunoprecipitated with an AP2 antibody was separated on SDS-PAGE, and Western blot was performed with an antibody against the Na+,K+-ATPase -subunit (left panel). Right panel, Quantitative analysis of three experiments. *P<0.05.

Presence of Hypertensive -Adducin Leads to Increased AP2-µ2 Phosphorylation
During endocytosis, AP2 undergoes phosphorylation in its µ2 subunit,^31,32 and this effect is required for its recognition of cargo molecules. Moreover, in cells expressing the AP2-µ2 T156A mutant (lacking the phosphorylation site), DA failed to promote Na⁺,K⁺-ATPase endocytosis and reduce Na⁺,K⁺-ATPase activity (Z. Chen, 2004, unpublished data). Thus, we next examined whether the presence of a mutant form of adducin would affect AP2 phosphorylation. Under nonstimulated conditions, phosphorylation of µ2 was significantly increased in cells transfected with F316Y-transfected compared with -WT transfected cells (Figure 3a). This effect was not observed with other proteins, such as the Na⁺,K⁺-ATPase, Na⁺/H⁺-exchanger (NH3), or DA receptor (data not shown). DA promoted an increase in µ2 phosphorylation in cells transiently transfected with the -WT. However, DA failed to elicit such effect in cells transfected with the F316Y (Figure 3b), likely because of the large quantities of AP2 µ2 molecules that already exist in phosphorylated form.

View larger version (25K): [in this window] [in a new window]   Figure 3. The state of AP2-µ2 phosphorylation was evaluated in OK cells transiently transfected with rat adducin -WT or F316Y. a, AP2-µ2 phosphorylation was calculated as the ratio of phosphorylated/immunoprecipitated AP2-µ2. A representative Western blot (top) and quantitative analysis (bottom) are shown; n=3. b, Phosphorylation of immunoprecipitated µ2 from OK cells incubated with DA (2 minutes). Representative Western blot (top) and quantitative analysis (bottom) are shown; n=5; *P<0.05 c, PPase specific activity was determined in 100 000g supernatants of nontransfected OK cells (NT), mock-transfected, and cells transfected with the rat (-WT) or hypertensive (F316Y); n=3. d, OK cells were transfected as in c with the different rat adducins carrying an HA tag. PPase activity (n=8) was determined in the immunoprecipitated material with an antibody against the HA tag. Immunoprecipitates from NT, mock, or those performed with a secondary antibody or the beads alone yielded no phosphatase activity. Data are expressed in pmol per minute because the protein in the immunoprecipitated material was not quantified. The amount of immunoprecipitated protein used for the PPase activity determinations was examined by Western blot. In addition, those samples were also probed with a PP2A antibody (Transduction Laboratories); (e) to identify the PPase isoform interacting with adducin. Left panel, Representative Western blot (WB); right panel, quantitative data of three experiments.

The phosphorylation state of a protein is determined by the balanced activity of protein kinases and PPase. Thus, we determined whether the presence of F316Y might alter PPase activity. Total PPase activity was the same in nontransfected OK cells, cells expressing the -WT, and cells expressing the -F316Y (Figure 3c). However, the immunoprecipitated material with HA antibody from cells expressing the -WT expresses PPase activity, and this activity was significantly reduced (35%) in immunoprecipitates from cells transfected with the -F316Y (Figure 3d). These observations suggest that PPase may directly or indirectly interact with adducin and that this interaction is impaired in cells expressing the hypertensive adducin. Further proof of an interaction between adducin and PPase was obtained by Western blot analysis. PPase 2A (PP2A) was coimmunoprecipitated with an HA antibody in cells transfected with the HA-tagged -WT (Figure 3e). Moreover, the amount of PP2A coimmunoprecipitated with HA-adducin was significantly reduced in cells transfected with the -F316Y, and this interaction was not affected by the presence of DA (supplemental Figure I, online data supplement). The total amount of immunoprecipitated HA-adducin was the same in both conditions (Figure 3e, bottom). A small amount of PPase 1 was also immunoprecipitated, and no difference was observed between cells expressing either form of adducin (data not shown). These results suggest that the normotensive adducin interacts directly or indirectly with PP2A and that this interaction is altered by the presence of the hypertensive variant. This defect might affect the location of a particular pool of phosphatase (linking AP2 dephosphorylation to its binding to the Na⁺,K⁺-ATPase -subunit) rather than affecting total cell PPase activity. We used confocal imaging to further evaluate the presence of a complex between the Na⁺,K⁺-ATPase -subunit, adducin, and PP2A. We observed colocalization between adducin and PP2A in OK cells expressing the normotensive adducin (Figure 4; normotensive, PP2A+adducin), whereas in cells expressing the hypertensive adducin, no evident colocalization was observed (Figure 4; hypertensive, PP2A+adducin). These results are consistent with the biochemical observation demonstrating an interaction of adducin and PP2A in cells expressing the wild-type but not the hypertensive adducin. Additionally, the results also demonstrated an interaction between the Na⁺,K⁺-ATPase -subunit with adducin or with the PP2A (as demonstrated previously³¹), and no differences could be seen between cells expressing the wild-type or the mutant form of adducin (data not shown). When all three images were merged, we could observe an association between the Na⁺,K⁺-ATPase -subunit, PP2A, and wild-type adducin, indicated by the white arrows and circle (merge-normotensive) but not with the mutant adducin (merge-hypertensive).

View larger version (127K): [in this window] [in a new window]   Figure 4. Confocal images of OK cells expressing the Na+,K+-ATPase -subunit (NK) bearing green fluorescent protein that were transiently transfected with the HA-adducin cDNA (normotensive or hypertensive). Bottom panels, Enlarged merged images (NK, adducin, and PP2A). Association of all three fluorophores (white) is denoted by arrows or white circle. This type of experiment was repeated on two separate occasions, and multiple cells in each experiment were examined.

Effects of the Hypertensive Variant of Human -Adducin on Na⁺,K⁺-ATPase Activity and Endocytosis
Because mutations in the human -adducin associated with high blood pressure are different from those present in the rat,¹ we determined whether they do exhibit comparable responses to DA (Figure 5). In OK cells transiently transfected with the human variant of adducin carrying the G460W/S586C hypertensive mutation, DA failed to promote -subunit endocytosis (Figure 5a) and consequently to reduce Na⁺,K⁺-ATPase activity, as opposed to its effect in -WT adducin–transfected cells (Figure 5b).

View larger version (19K): [in this window] [in a new window]   Figure 5. Effect of human adducin during DA-mediated effects on Na+,K+-ATPase activity and endocytosis. OK cells were transiently transfected with the human -WT adducin or G460W/S586C variant. Na+,K+-ATPase -subunit abundance (cell surface biotinylation) in the plasma membrane (a) and Na+,K+-ATPase activity (nmol rubidium/mg protein per minute; b) were determined in intact cells incubated previously with vehicle or DA (both n=3). *P<0.05

The effect of hypertensive adducin seemed to be confined to the Na⁺,K⁺-ATPase. We have demonstrated previously that changes in intracellular sodium affect the distribution of DA (DA1r) and angiotensin II (AT1r) receptors within the plasma membrane.³² Neither the plasma membrane content of DA1r and AT1r nor their distribution in response to high intracellular sodium was affected by the hypertensive adducin (supplemental Figure II, online supplement data).

Hypertensive Variant of Human -Adducin Is Associated With Higher Na⁺,K⁺-ATPase Activity and Increased Pool of Active Molecules at the Plasma Membrane
Transient expression of the hypertensive variant of human adducin in OK cells resulted in higher basal Na⁺,K⁺-ATPase activity (WT 8.1±0.3 versus G460W/S586C 9.7±0.5; both n=3; P<0.05; Figure 6a). A controlled increase in intracellular sodium (induced by monensin)²³ elicited a larger increase in Na⁺,K⁺-ATPase activity in cells expressing the G460W/S586C (in percent 104±2.2; n=3) compared with -WT (in percent 87±7.5; n=3). Additionally, an increase in Na⁺,K⁺-ATPase -subunit abundance at the plasma membrane is observed in cells expressing the G460W/S586C when compared with the -WT (Figure 6b). Similar to Milan rats, an increase in Na⁺, K⁺-ATPase abundance has also been reported in PCTs from spontaneously hypertensive rats.³³ Moreover, increases in intracellular sodium (induced by monensin) are associated with higher Na⁺,K⁺-ATPase -subunit at the plasma membrane of cells expressing only the G460W/S586C. These results suggest that the increase in basal Na⁺,K⁺-ATPase activity associated with the presence of the hypertensive adducin is attributable to a higher number of Na⁺,K⁺-ATPase molecules within the plasma membrane, whereas the increased catalytic activity in response to monensin (high sodium) could be the result of an increased catalytic activity and also possibly an additional increase in active molecules at the plasma membrane.

View larger version (26K): [in this window] [in a new window]   Figure 6. Effect of high intracellular sodium on Na+,K+-ATPase activity and -subunit distribution of the plasma membrane in OK cells transiently expressing the human -WT or G460W/S586C. a, Na+,K+-ATPase activity in nontransfected, -WT-transfected or G460W/S586C-transfected cells treated with (high sodium) or without (normal sodium) monensin (30 minutes at room temperature) (n=4; triplicate determinations). b, Na+,K+-ATPase -subunit abundance at the plasma membrane (cell surface biotinylation) under the same conditions described in a. Representative Western blot and quantitative analysis of three experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study demonstrates a defective endocytic process of Na⁺,K⁺-ATPase units from the plasma membrane that is associated with the presence of hypertension-linked mutations in the -adducin molecule. Using DA as the prototype GPCR (G-protein–coupled receptor) agonist (and also an important agent in the regulation of renal sodium excretion during salt loading),¹¹ we found that DA failed to promote the endocytosis of active Na⁺,K⁺-ATPase molecules and to decrease Na⁺,K⁺-ATPase activity. This was observed in isolated PCTs obtained from MHS rats, as well as in a renal cell line expressing the rat or human hypertensive variant of adducin. The cellular defect whereby adducin prevents Na⁺,K⁺-ATPase endocytosis in response to DA occurs at a later stage of the endocytic signal, namely the phosphorylation and recruitment of AP2 and its association with the Na⁺,K⁺-ATPase -subunit, whereas earlier stages of this cascade (reflecting DA receptor signaling), including phosphorylation of the Na⁺,K⁺-ATPase -subunit and stimulation of PI3K, are unaffected.

The possibility of a defect within the Na⁺,K⁺-ATPase itself as the cause of the impaired endocytosis observed in OK cells transiently expressing the different hypertensive forms of adducin seems excluded because the only variable introduced was the expression of such adducin mutants. For the same reason, it is also possible to rule out an impaired response to DA because of a native abnormal DA receptor–G-protein denylyl cyclase coupling, as suggested previously for another rat model of essential hypertension.^34,35 Thus, one can speculate that the defect responsible for deficient endocytosis might be located at the plasma membrane interface between the Na⁺,K⁺-ATPase and the endocytic network.

Clathrin-dependent endocytosis of membrane proteins is initiated by adaptor proteins (adaptins) that simultaneously bind to cargo proteins, recruit clathrin, and promote the formation of clathrin-coated pits.³⁶ In the present study, DA promoted the association of AP2 with the Na⁺,K⁺-ATPase -subunit in renal cells isolated from normotensive rats but not from hypertensive rats or in cells expressing transiently the rat or human hypertensive adducin. AP2 phosphorylation is a key event that determines the binding affinity of the µ2 subunit to cargo proteins.^29,30,37 Interestingly, the basal (nonstimulated) state of phosphorylation of AP2-µ2 is higher in cells transfected with the hypertensive F316Y adducin, although they showed reduced endocytosis, suggesting that it is the transition between phospho/dephospo–AP2-µ2 that facilitates its association with the cargo molecules, as demonstrated by previous in vitro studies.³⁷

At present, it is unclear how a mutated form of adducin translates into altered AP2-µ2 subunit phosphorylation and Na⁺,K⁺-ATPase endocytosis. The present data suggest the possibility that a PPase associates directly or as part of a complex with adducin, and that this association with the hypertensive adducin variant is absent, affecting its ability to dephosphorylate the AP2 subunit within the Na⁺,K⁺-ATPase microdomain (supplemental Figure III, online data supplement) but not the traffic of other proteins such as DA and AT2 receptors (supplemental Figure II, online data supplement).

An increase in basal (nonstimulated) Na⁺,K⁺-ATPase activity (between 20% and 35%) is observed in PCT cells derived from MHS rats and in cells transiently transfected with the hypertensive form of either rat or human adducin. This increase in activity is associated with an increase in the amount of Na⁺,K⁺-ATPase molecules present at the plasma membrane. A similar increase in Na⁺, K⁺-ATPase abundance has been reported in PCT cells from spontaneously hypertensive rats.³³ At present, the causes of this effect are not evident. Our data in transiently transfected cells point to a short-term influence of higher intracellular sodium on Na⁺,K⁺-ATPase translocation to the plasma membrane combined with a higher catalytic activity. Whereas the in vitro studies in OK cells may exclude an increase in Na⁺,K⁺-ATPase synthesis, this pathway may likely contribute to a higher activity in Milan rats and humans. It is possible that the previously described¹⁰ stiff cortical actin cytoskeleton lacking a dynamic regulation (as a direct consequence of a mutated adducin) may affect the endocytic rate and increase the time that the Na⁺,K⁺-ATPase molecules reside at the plasma membrane. Other membrane proteins, such as the distribution of DA1 and AT1 receptors, are not affected by the hypertensive form of adducin; the reasons why this phenomenon appears to be segregated to the Na⁺,K⁺-ATPase molecules remain unclear.

Subjects carrying another hypertensive mutation (-adducin 460W variant) display a faster proximal tubular sodium reabsorption (measured as lithium clearance) and transport across the erythrocyte membrane,¹ greater blood pressure response to saline infusion, lower plasma renin, and greater response to diuretics compared with individuals carrying the normotensive -adducin variant.^1–3 These observations are consistent with a constitutive increase in sodium transport rate across tubular cells and are paralleled by data at the biochemical level, whereas in a cell-free system, the rat F316Y and the human G460W display a greater ATP affinity during stimulation of Na⁺,K⁺-ATPase than the normotensive variants.³⁸ Our studies performed in a cell system also reveal that the similarities in responses of rats and humans occur despite the differences in amino acids that are mutated between the two species.

In summary, the present data indicate that in certain types of hypertensive animal models (eg, the Milan rat strain), a deficient endocytosis of Na⁺,K⁺-ATPase molecules might be an important contributing factor for the increased capacity of the renal tubule cells to reabsorb sodium, thus promoting sodium retention and the development of high blood pressure. The results could also furnish a biochemical working hypothesis for explaining recent data in humans, demonstrating that the mutations occurring in the adducin molecule may increase sodium retention, with the risk of developing hypertension and its associated cardiovascular and renal complications.

	Acknowledgments

 
This work was supported in part with funds from the Swedish Research Council (10860), the Swedish Heart and Lung Foundation, and the National Institutes of Health (DK062195 and DK53460). We thank L. Torielli and P. Ferrari for critical reading of this manuscript. The technical help of Marie-Odile Revel is appreciated.

	Footnotes

 
Original received July 19, 2004; revision received October 21, 2004; accepted October 25, 2004.

	References

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