Carlos H. Pedemonte

Hypoxia-induced endocytosis of Na,K-ATPase in alveolar epithelial cells is mediated by mitochondrial reactive oxygen species and PKC-ζ

During ascent to high altitude and pulmonary edema, the alveolar epithelial cells (AEC) are exposed to hypoxic conditions. Hypoxia inhibits alveolar fluid reabsorption and decreases Na,K-ATPase activity in AEC. We report here that exposure of AEC to hypoxia induced a time-dependent decrease of Na,K-ATPase activity and a parallel decrease in the number of Na,K-ATPase alpha(1) subunits at the basolateral membrane (BLM), without changing its total cell protein abundance. These effects were reversible upon reoxygenation and specific, because the plasma membrane protein GLUT1 did not decrease in response to hypoxia. Hypoxia caused an increase in mitochondrial reactive oxygen species (ROS) levels that was inhibited by antioxidants. Antioxidants prevented the hypoxia-mediated decrease in Na,K-ATPase activity and protein abundance at the BLM. Hypoxia-treated AEC deficient in mitochondrial DNA (rho(0) cells) did not have increased levels of ROS, nor was the Na,K-ATPase activity inhibited. Na,K-ATPase alpha(1) subunit was phosphorylated by PKC in hypoxia-treated AEC. In AEC treated with a PKC-zeta antagonist peptide or with the Na,K-ATPase alpha(1) subunit lacking the PKC phosphorylation site (Ser-18), hypoxia failed to decrease Na,K-ATPase abundance and function. Accordingly, we provide evidence that hypoxia decreases Na,K-ATPase activity in AEC by triggering its endocytosis through mitochondrial ROS and PKC-zeta-mediated phosphorylation of the Na,K-ATPase alpha(1) subunit.

Phosphorylation of the catalyic alpha-subunit constitutes a triggering signal for Na+,K+-ATPase endocytosis.

Inhibition of Na+,K+-ATPase activity by dopamine is an important mechanism by which renal tubules modulate urine sodium excretion during a high salt diet. However, the molecular mechanisms of this regulation are not clearly understood. Inhibition of Na+,K+-ATPase activity in response to dopamine is associated with endocytosis of its α- and β-subunits, an effect that is protein kinase C-dependent. In this study we used isolated proximal tubule cells and a cell line derived from opossum kidney and demonstrate that dopamine-induced endocytosis of Na+,K+-ATPase and inhibition of its activity were accompanied by phosphorylation of the α-subunit. Inhibition of both the enzyme activity and its phosphorylation were blocked by the protein kinase C inhibitor bisindolylmaleimide. The early time dependence of these processes suggests a causal link between phosphorylation and inhibition of enzyme activity. However, after 10 min of dopamine incubation, the α-subunit was no longer phosphorylated, whereas enzyme activity remained inhibited due to its removal from the plasma membrane. Dephosphorylation occurred in the late endosomal compartment. To further examine whether phosphorylation was a prerequisite for subunit endocytosis, we used the opossum kidney cell line transfected with the rodent α-subunit cDNA. Treatment of this cell line with dopamine resulted in phosphorylation and endocytosis of the α-subunit with a concomitant decrease in Na+,K+-ATPase activity. In contrast, none of these effects were observed in cells transfected with the rodent α-subunit that lacks the putative protein kinase C-phosphorylation sites (Ser11 and Ser18). Our results support the hypothesis that protein kinase C-dependent phosphorylation of the α-subunit is essential for Na+,K+-ATPase endocytosis and that both events are responsible for the decreased enzyme activity in response to dopamine.

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

&agr;-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-&mgr;2 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, &mgr;2-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.

PKC-β and PKC-ζ mediate opposing effects on proximal tubule Na+,K+-ATPase activity

Dopamine (DA) inhibits rodent proximal tubule Na+,K+‐ATPase via stimulation of protein kinase C (PKC). However, direct stimulation of PKC by phorbol 12‐myristate 13‐acetate (PMA) results in increased Na+,K+‐ATPase. LY333531, a specific inhibitor of the PKC‐β isoform, prevents PMA‐dependent activation of Na+,K+‐ATPase, but has no effect on DA inhibition of this activity. A similar result was obtained with a PKC‐β inhibitor peptide. Concentrations of staurosporine, that inhibits PKC‐ζ, prevent DA‐dependent inhibition of Na+,K+‐ATPase and a similar effect was obtained with a PKC‐ζ inhibitor peptide. Thus, PMA‐dependent stimulation of Na+,K+‐ATPase is mediated by activation of PKC‐β, whereas inhibition by DA requires activation of PKC‐ζ.

Tyrosine 537 within the Na+,K+-ATPase α-Subunit Is Essential for AP-2 Binding and Clathrin-dependent Endocytosis

In renal epithelial cells endocytosis of Na+,K+-ATPase molecules is initiated by phosphorylation of its α1-subunit, leading to activation of phosphoinositide 3-kinase and adaptor protein-2 (AP-2)/clathrin recruitment. The present study was performed to establish the identity of the AP-2 recognition domain(s) within the Na+,K+-ATPase α1-subunit. We identified a conserved sequence (Y537LEL) within the α1-subunit that represents an AP-2 binding site. Binding of AP-2 to the Na+,K+-ATPase α1-subunit in response to dopamine (DA) was increased in OK cells stably expressing the wild type rodent α-subunit (OK-WT), but not in cells expressing the Y537A mutant (OK-Y537A). DA treatment was associated with increased α1-subunit abundance in clathrin vesicles from OK-WT but not from OK-Y537A cells. In addition, this mutation also impaired the ability of DA to inhibit Na+,K+-ATPase activity. Because phorbol estersincrease Na+,K+-ATPase activity in OK cells, and this effect was not affected by the Y537A mutation, the present results suggest that the identified motif is specifically required for DA-induced AP-2 binding and Na+,K+-ATPase endocytosis.

Regulation of Na,K-ATPase transport activity by protein kinase C.

Abstract. Considerable evidence indicates that the renal Na+,K+-ATPase is regulated through phosphorylation/dephosphorylation reactions by kinases and phosphatases stimulated by hormones and second messengers. Recently, it has been reported that amino acids close to the NH2-terminal end of the Na+,K+-ATPase α-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na+,K+-ATPase activity. To determine whether the α-subunit NH2-terminus is involved in the regulation of Na+,K+-ATPase activity by PKC, we have expressed the wild-type rodent Na+,K+-ATPase α-subunit and a mutant of this protein that lacks the first thirty-one amino acids at the NH2-terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant phenotype characteristic of rodent kidney cells. The presence of the α-subunit NH2-terminal segment was not necessary to express the maximal Na+,K+-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb+-transport in intact cells were the same in cells transfected with the wild-type rodent α1 and the NH2-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na+,K+-ATPase mediated Rb+-uptake and reduced the intracellular Na+ concentration of cells transfected with wild-type α1 cDNA. In contrast, these effects were not observed in cells expressing the NH2-deletion mutant of the α-subunit. Treatment with phorbol ester appears to affect specifically the Na+,K+-ATPase activity and no evidence was observed that other proteins involved in Na+-transport were affected. These results indicate that amino acid(s) located at the α-subunit NH2-terminus participate in the regulation of the Na+,K+-ATPase activity by PKC.

Agonist-dependent Regulation of Renal Na+,K+-ATPase Activity Is Modulated by Intracellular Sodium Concentration

We tested the hypothesis that the level of intracellular sodium modulates the hormonal regulation of the Na+,K+-ATPase activity in proximal tubule cells. By using digital imaging fluorescence microscopy of a sodium-sensitive dye, we determined that the sodium ionophore monensin induced a dose-specific increase of intracellular sodium. A correspondence between the elevation of intracellular sodium and the level of dopamine-induced inhibition of Na+,K+-ATPase activity was determined. At basal intracellular sodium concentration, stimulation of cellular protein kinase C by phorbol 12-myristate 13-acetate (PMA) promoted a significant increase in Na+,K+-ATPase activity; however, this activation was gradually reduced as the concentration of intracellular sodium was increased to become a significant inhibition at concentrations of intracellular sodium higher than 16 mm. Under these conditions, PMA and dopamine share the same signaling pathway to inhibit the Na+,K+-ATPase. The effects of PMA and dopamine on the Na+,K+-ATPase activity and the modulation of these effects by different intracellular sodium concentrations were not modified when extracellular and intracellular calcium were almost eliminated. These results suggest that the level of intracellular sodium modulates whether hormones stimulate, inhibit, or have no effect on the Na+,K+-ATPase activity leading to a tight control of sodium reabsorption.

Hormonal‐dependent recruitment of Na+,K+‐ATPase to the plasmalemma is mediated by PKCβ and modulated by [Na+]i

The present study demonstrates that stimulation of hormonal receptors of proximal tubule cells with the serotonin‐agonist 8‐hydroxy‐2‐(di‐n‐propylamino) tetraline (8‐OH‐DPAT) induces an augmentation of Na+,K+‐ATPase activity that results from the recruitment of enzyme molecules to the plasmalemma. Cells expressing the rodent wild‐type Na+,K+‐ATPase α‐subunit had the same basal Na+,K+‐ATPase activity as cells expressing the α‐subunit S11A or S18A mutants, but stimulation of Na+,K+‐ATPase activity was completely abolished in either mutant. 8‐OH‐DPAT treatment of OK cells led to PKCβ‐dependent phosphorylation of the α‐subunit Ser‐11 and Ser‐18 residues, and determination of enzyme activity with the S11A and S18A mutants indicated that both residues are essential for the agonist‐dependent stimulation of Na+,K+‐ATPase activity. When cells were treated with both dopamine and 8‐OH‐DPAT, an activation of Na+,K+‐ATPase was observed at basal intracellular sodium concentration (∼9 mM), and this activation was gradually reduced and became a significant inhibition as the concentration of intracellular sodium gradually increased from 9 to 19 mM. Thus, besides the antagonistic effects of dopamine and 8‐OH‐DPAT, intracellular sodium modulates whether an activation or an inhibition of Na+,K+‐ATPase is produced.

Trafficking of Na-K-ATPase and dopamine receptor molecules induced by changes in intracellular sodium concentration of renal epithelial cells.

Most of the transepithelial transport of sodium in proximal tubules occurs through the coordinated action of the apical sodium/proton exchanger and the basolateral Na-K-ATPase. Hormones that regulate proximal tubule sodium excretion regulate the activities of these proteins. We have previously demonstrated that the level of intracellular sodium concentration modulates the regulation of Na-K-ATPase activity by angiotensin II and dopamine. An increase of a few millimolars in intracellular sodium concentration leads to increased Na-K-ATPase activity without a statistically significant increase in the number of plasma membrane Na-K-ATPase molecules, as determined by cell surface protein biotinylation. Using total internal reflection fluorescence, we detected an increased number of Na-K-ATPase molecules in cytosolic compartments adjacent to the plasma membrane, suggesting that the increased intracellular sodium concentration induces a movement of Na-K-ATPase molecules toward the plasma membrane. While intracellular compartments containing Na-K-ATPase molecules are very close to the plasma membrane, compartments containing type 1 dopamine receptors (D1Rs) are distributed in different parts of the cell cytosol. Fluorescence determinations indicate that an increased intracellular sodium concentration induces the increased colocalization of dopamine receptors with Na-K-ATPase molecules in the region of the plasma membrane. We propose that under in vivo conditions, in response to a sodium load in the lumen of proximal tubules, an increased level of intracellular sodium in epithelial cells is an early event that triggers the cellular response that leads to dopamine inhibition of proximal tubule sodium reabsorption.

Contrary to Rat-Type, Human-Type Na,K-ATPase Is Phosphorylated at the Same Amino Acid by Hormones that Produce Opposite Effects on Enzyme Activity

Renal sodium homeostasis is a major determinant of BP and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular secondary messengers, either activate or inhibit proximal tubule Na,K-ATPase. It was shown previously that phorbol esters and angiotensin II and serotonin induce the phosphorylation of both Ser-11 and Ser-18 of the Na,K-ATPase alpha-subunit. This results in the recruitment of Na,K-ATPase molecules to the plasma membrane and an increased capacity to transport sodium ions. Treatment of the same cells with dopamine leads to phosphorylation of the Na,K-ATPase alpha-subunit Ser-18. The subsequent internalization of Na,K-ATPase molecules results in a reduced capacity to transport sodium ions. These effects are observed in cells that express the rat-type Na,K-ATPase. However, the Na,K-ATPase alpha1-subunit of several species, such as human, pig, and mouse, does not have a Ser-18 in their N-terminal region. Therefore, the possibility exists that, in those species, the Na,K-ATPase is not regulated by the hormones that regulate natriuresis. This study presents evidence that in cells that express the human-type Na,K-ATPase, dopamine inhibits and phorbol esters activate the Na,K-ATPase-mediated transport. These opposite effects are mediated by the phosphorylation of the same amino acid residue, Ser-11 of Na,K-ATPase alpha1, and the presence of alpha1 Ser-18 is not essential for the hormonal regulation of Na,K-ATPase activity in LLCPK1 cells. It was observed that, whereas the regulatory stimulation of Na,K-ATPase is mediated by protein kinase Cbeta, the regulatory inhibition is mediated by protein kinase Czeta. This is similar to what was demonstrated previously in cells that express the rat-type Na,K-ATPase.