Pals-associated tight junction protein functionally links dopamine and angiotensin II to the regulation of sodium transport in renal epithelial cells.

BACKGROUND AND PURPOSE: Dopamine inhibits renal cell Na(+),K(+)-ATPase activity and cell sodium transport by promoting the internalization of active molecules from the plasma membrane, whereas angiotensin II (ATII) stimulates its activity by recruiting new molecules to the plasma membrane. They achieve such effects by activating multiple and distinct signalling molecules in a hierarchical manner. The purpose of this study was to investigate whether dopamine and ATII utilize scaffold organizer proteins as components of their signalling networks, in order to avoid deleterious cross talk. EXPERIMENTAL APPROACH: Attention was focused on a multiple PDZ domain protein, Pals-associated tight junction protein (PATJ). Ectopic expression of PATJ in renal epithelial cells in culture was used to study its interaction with components of the dopamine signalling cascade. Similarly, expression of PATJ deletion mutants was employed to analyse its functional relevance during dopamine-, ATII- and insulin-dependent regulation of Na(+),K(+)-ATPase. KEY RESULTS: Dopamine receptors and components of its signalling cascade mediating inhibition of Na(+),K(+)-ATPase interact with PATJ. Inhibition of Na(+),K(+)-ATPase by dopamine was prevented by expression of mutants of PATJ lacking PDZ domains 2, 4 or 5; whereas the stimulatory effect of ATII and insulin on Na(+),K(+)-ATPase was blocked by expression of PATJ lacking PDZ domains 1, 4 or 5. CONCLUSIONS AND IMPLICATIONS: A multiple PDZ domain protein may add functionality to G protein-coupled and tyrosine kinase receptors signalling during regulation of Na(+),K(+)-ATPase. Signalling molecules and effectors can be integrated into a functional network by the scaffold organizer protein PATJ via its multiple PDZ domains.

Localization of intracellular compartments that exchange Na,K-ATPase molecules with the plasma membrane in a hormone-dependent manner.

BACKGROUND AND PURPOSE: Dopamine is a major regulator of sodium reabsorption in proximal tubule epithelia. By binding to D1-receptors, dopamine induces endocytosis of plasma membrane Na,K-ATPase, resulting in a reduced capacity of the cells to transport sodium, thus contributing to natriuresis. We have previously demonstrated several aspects of the molecular mechanism by which dopamine induces Na,K-ATPase endocytosis; however, the location of intracellular compartments containing Na,K-ATPase molecules has not been identified. EXPERIMENTAL APPROACH: In this study, we used different approaches to determine the localization of Na,K-ATPase-containing intracellular compartments. By expression of fluorescent-tagged Na,K-ATPase molecules in opossum kidney cells, a cell culture model of proximal tubule epithelia, we used fluorescence microscopy to determine cellular distribution of the fluorescent molecules and the effects of dopamine on this distribution. By labelling cell surface Na,K-ATPase molecules from the cell exterior with either biotin or an epitope-tagged antibody, we determined the localization of the tagged Na,K-ATPase molecules after endocytosis induced by dopamine. KEY RESULTS: In cells expressing fluorescent-tagged Na,K-ATPase molecules, there were intracellular compartments containing Na,K-ATPase molecules. These compartments were in very close proximity to the plasma membrane. Upon treatment of the cells with dopamine, the fluorescence labelling of these compartments was increased. The labelling of these compartments was also observed when the endocytosis of biotin- or antibody-tagged plasma membrane Na,K-ATPase molecules was induced by dopamine. CONCLUSIONS AND IMPLICATIONS: The intracellular compartments containing Na,K-ATPase molecules are located just underneath the plasma membrane.

Altered dopaminergic transmission in the anorexic anx/anx mouse striatum.

We demonstrate abnormal dopaminergic neurotransmission in anorexic mice, homozygous for a recessive mutation (anx) causing starvation and motor disturbances. Isolated neurons from anx/anx striatum displayed a markedly increased activity of the Na+,K+-ATPase compared with normal littermates. Dopamine down-regulates Na+,K+-ATPase activity in striatal medium spiny neurons in rat, mouse and guinea pig. However, addition of dopamine in vitro failed to suppress the increased activity in anx/anx striatal neurons. Striatal dopamine and its metabolites, but not norepinephrine, were slightly but significantly lower in anx/anx mice than in normal littermates. We suggest that abnormal dopaminergic transmission may contribute to the anx phenotype.

Short-term regulation of the proximal tubule Na+,K+-ATPase: increased/decreased Na+,K+-ATPase activity mediated by protein kinase C isoforms.

In different species and tissues, a great variety of hormones modulate Na+,K+-ATPase activity in a short-term fashion. Such regulation involves the activation of distinct intracellular signaling networks that are often hormone- and tissue-specific. This minireview focuses on our own experimental observations obtained by studying the regulation of the rodent proximal tubule Na+,K+-ATPase. We discuss evidence that hormones responsible for regulating kidney proximal tubule sodium reabsorption may not affect the intrinsic catalytic activity of the Na+,K+-ATPase, but rather the number of active units within the plasma membrane due to shuttling Na+,K+-ATPase molecules between intracellular compartments and the plasma membrane. These processes are mediated by different isoforms of protein kinase C and depend largely on variations in intracellular sodium concentrations.

Simultaneous phosphorylation of Ser11 and Ser18 in the alpha-subunit promotes the recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane.

Renal sodium homeostasis is a major determinant of blood pressure and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular second messengers, either activate or inhibit proximal tubule Na(+),K(+)-ATPase. We have shown previously that phorbol ester (PMA) stimulation of endogenous PKC leads to activation of Na(+),K(+)-ATPase in cultured proximal tubule cells (OK cells) expressing the rodent Na(+), K(+)-ATPase alpha-subunit. We have now demonstrated that the treatment with PMA leads to an increased amount of Na(+),K(+)-ATPase molecules in the plasmalemma, which is proportional to the increased enzyme activity. Colchicine, dinitrophenol, and potassium cyanide prevented the PMA-dependent stimulation of activity without affecting the increased level of phosphorylation of the Na(+), K(+)-ATPase alpha-subunit. This suggests that phosphorylation does not directly stimulate Na(+),K(+)-ATPase activity; instead, phosphorylation may be the triggering mechanism for recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane. Transfected cells expressing either an S11A or S18A mutant had the same basal Na(+),K(+)-ATPase activity as cells expressing the wild-type rodent alpha-subunit, but PMA stimulation of Na(+),K(+)-ATPase activity was completely abolished in either mutant. PMA treatment led to phosphorylation of the alpha-subunit by stimulation of PKC-beta, and the extent of this phosphorylation was greatly reduced in the S11A and S18A mutants. These results indicate that both Ser11 and Ser18 of the alpha-subunit are essential for PMA stimulation of Na(+), K(+)-ATPase activity, and that these amino acids are phosphorylated during this process. The results presented here support the hypothesis that PMA regulation of Na(+),K(+)-ATPase is the result of an increased number of Na(+),K(+)-ATPase molecules in the plasma membrane.

Phosphoinositide-3 kinase binds to a proline-rich motif in the Na+, K+-ATPase alpha subunit and regulates its trafficking.

Endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor stimulation requires activation of class I(A) phosphoinositide-3 kinase (PI3K-I(A)) in a protein kinase C-dependent manner. In this paper, we report that PI3K-I(A), through its p85alpha subunit-SH3 domain, binds to a proline-rich region in the Na(+),K(+)-ATPase catalytic alpha subunit. This interaction is enhanced by protein kinase C-dependent phosphorylation of a serine residue that flanks the proline-rich motif in the Na(+),K(+)-ATPase alpha subunit and results in increased PI3K-I(A) activity, an effect necessary for adaptor protein 2 binding and clathrin recruitment. Thus, Ser-phosphorylation of the Na(+),K(+)-ATPase catalytic subunit serves as an anchor signal for regulating the location of PI3K-I(A) and its activation during Na(+),K(+)-ATPase endocytosis in response to G protein-coupled receptor signals.

G protein-coupled receptors regulate Na+,K+-ATPase activity and endocytosis by modulating the recruitment of adaptor protein 2 and clathrin.

Inhibition of Na(+),K(+)-ATPase (NKA) activity in renal epithelial cells by activation of G protein-coupled receptors is mediated by phosphorylation of the catalytic alpha-subunit followed by endocytosis of active molecules. We examined whether agonists that counteract this effect do so by dephosphorylation of the alpha-subunit or by preventing its internalization through a direct interaction with the endocytic network. Oxymetazoline counteracted the action of dopamine on NKA activity, and this effect was achieved not by preventing alpha-subunit phosphorylation, but by impaired endocytosis of alpha-subunits into clathrin vesicles and early and late endosomes. Dopamine-induced inhibition of NKA activity and alpha-subunit endocytosis required the interaction of adaptor protein 2 (AP-2) with the catalytic alpha-subunit. Phosphorylation of the alpha-subunit is essential because dopamine failed to promote such interaction in cells lacking the protein kinase C phosphorylation residue (S18A). Confocal microscopy confirmed that oxymetazoline prevents incorporation of NKA molecules into clathrin vesicles by inhibiting the ability of dopamine to recruit clathrin to the plasma membrane. Dopamine decreased the basal levels of inositol hexakisphosphate (InsP(6)), whereas oxymetazoline prevented this effect. Similar increments (above basal) in the concentration of InsP(6) induced by oxymetazoline prevented AP-2 binding to the NKA alpha-subunit in response to dopamine. In conclusion, inhibition of NKA activity can be reversed by preventing its endocytosis without altering the state of alpha-subunit phosphorylation; increased InsP(6) in response to G protein-coupled receptor signals blocks the recruitment of AP-2 and thereby clathrin-dependent endocytosis of NKA.

Syntaxin 1 interacts with the L(D) subtype of voltage-gated Ca(2+) channels in pancreatic beta cells.

Interaction of syntaxin 1 with the alpha(1D) subunit of the voltage-gated L type Ca(2+) channel was investigated in the pancreatic beta cell. Coexpression of the enhanced green fluorescent protein-linked alpha(1D) subunit with the enhanced blue fluorescent protein-linked syntaxin 1 and Western blot analysis together with subcellular fractionation demonstrated that the alpha(1D) subunit and syntaxin 1 were colocalized in the plasma membrane. Furthermore, the alpha(1D) subunit was coimmunoprecipitated efficiently by a polyclonal antibody against syntaxin 1. Syntaxin 1 also played a central role in the modulation of L type Ca(2+) channel activity because there was a faster Ca(2+) current run-down in cells incubated with antisyntaxin 1 compared with controls. In parallel, antisyntaxin 1 markedly reduced insulin release in both intact and permeabilized cells, subsequent to depolarization with K(+) or exposure to high Ca(2+). Exchanging Ca(2+) for Ba(2+) abolished the effect of antisyntaxin 1 on both Ca(2+) channel activity and insulin exocytosis. Moreover, antisyntaxin 1 had no significant effects on Ca(2+)-independent insulin release trigged by hypertonic stimulation. This suggests that there is a structure-function relationship between the alpha(1D) subunit of the L type Ca(2+) channel and the exocytotic machinery in the pancreatic beta cell.

Stimulation of the dopamine 1 receptor increases lung edema clearance.

We previously reported that lung edema clearance was stimulated by dopamine (DA). The purpose of this study was to determine whether the DA-mediated stimulation of edema clearance occurs via an adrenergic or dopaminergic regulation of alveolar epithelial Na, K-ATPase. When isolated perfused rat lungs were coinstilled with DA and SCH 23390 (a specific D(1) receptor antagonist), there was a dose-dependent attenuation of the stimulatory effects of DA. Coinstillation with S-sulpiride (a specific D(2) receptor antagonist) or propranolol (a beta-adrenergic antagonist) did not alter DA-stimulated clearance. Similarly, the specific dopaminergic D(1) agonist fenoldopam increased lung edema clearance, but quinpirole (a specific dopaminergic D(2) agonist) did not. (125)I-SCH 23982 binding studies suggested that D(1) receptors are expressed on alveolar type II (ATII) cells with an apparent dissociation constant (K(d)) of 4.4 nM and binding maximum (Bmax) 9.8 pmol/mg. Consistent with these results, the D(1) receptor messenger RNA (mRNA) and protein were detected in ATII cells by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. These data demonstrate a novel mechanism involving the activation of dopaminergic D(1) receptors which mediates DA-stimulated edema removal from rat lungs.

PKC-beta and PKC-zeta 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-beta 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-beta inhibitor peptide. Concentrations of staurosporine, that inhibits PKC-zeta, prevent DA-dependent inhibition of Na+,K+-ATPase and a similar effect was obtained with a PKC-zeta inhibitor peptide. Thus, PMA-dependent stimulation of Na+,K+-ATPase is mediated by activation of PKC-beta, whereas inhibition by DA requires activation of PKC-zeta.

Dopamine-induced endocytosis of Na+,K+-ATPase is initiated by phosphorylation of Ser-18 in the rat alpha subunit and Is responsible for the decreased activity in epithelial cells.

Dopamine inhibits Na+,K+-ATPase activity in renal tubule cells. This inhibition is associated with phosphorylation and internalization of the alpha subunit, both events being protein kinase C-dependent. Studies of purified preparations, fusion proteins with site-directed mutagenesis, and heterologous expression systems have identified two major protein kinase C phosphorylation residues (Ser-11 and Ser-18) in the rat alpha1 subunit isoform. To identify the phosphorylation site(s) that mediates endocytosis of the subunit in response to dopamine, we have performed site-directed mutagenesis of these residues in the rat alpha1 subunit and expressed the mutated forms in a renal epithelial cell line. Dopamine inhibited Na+,K+-ATPase activity and increased alpha subunit phosphorylation and clathrin-dependent endocytosis into endosomes in cells expressing the wild type alpha1 subunit or the S11A alpha1 mutant, and both effects were blocked by protein kinase C inhibition. In contrast, dopamine did not elicit any of these effects in cells expressing the S18A alpha1 mutant. While Ser-18 phosphorylation is necessary for endocytosis, it does not affect per se the enzymatic activity: preventing endocytosis with wortmannin or LY294009 blocked the inhibitory effect of dopamine on Na+,K+-ATPase activity, although it did not alter the increased alpha subunit phosphorylation induced by this agonist. We conclude that dopamine-induced inhibition of Na+, K+-ATPase activity in rat renal tubule cells requires endocytosis of the alpha subunit into defined intracellular compartments and that phosphorylation of Ser-18 is essential for this process.

Glucose decreases Na+,K+-ATPase activity in pancreatic beta-cells. An effect mediated via Ca2+-independent phospholipase A2 and protein kinase C-dependent phosphorylation of the alpha-subunit.

In the pancreatic beta-cell, glucose-induced membrane depolarization promotes opening of voltage-gated L-type Ca2+ channels, an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i), and exocytosis of insulin. Inhibition of Na+,K+-ATPase activity by ouabain leads to beta-cell membrane depolarization and Ca2+ influx. Because glucose-induced beta-cell membrane depolarization cannot be attributed solely to closure of ATP-regulated K+ channels, we investigated whether glucose regulates other transport proteins, such as the Na+,K+-ATPase. Glucose inhibited Na+,K+-ATPase activity in single pancreatic islets and intact beta-cells. This effect was reversible and required glucose metabolism. The inhibitory action of glucose was blocked by pretreatment of the islets with a selective inhibitor of a Ca2+-independent phospholipase A2. Arachidonic acid, the hydrolytic product of this phospholipase A2, also inhibited Na+, K+-ATPase activity. This effect, like that of glucose, was blocked by nordihydroguaiaretic acid, a selective inhibitor of the lipooxygenase metabolic pathway, but not by inhibitors of the cyclooxygenase or cytochrome P450-monooxygenase pathways. The lipooxygenase product 12(S)-HETE (12-S-hydroxyeicosatetranoic acid) inhibited Na+,K+-ATPase activity, and this effect, as well as that of glucose, was blocked by bisindolylmaleimide, a specific protein kinase C inhibitor. Moreover, glucose increased the state of alpha-subunit phosphorylation by a protein kinase C-dependent process. These results demonstrate that glucose inhibits Na+, K+-ATPase activity in beta-cells by activating a distinct intracellular signaling network. Inhibition of Na+,K+-ATPase activity may thus be part of the mechanisms whereby glucose promotes membrane depolarization, an increase in [Ca2+]i, and thereby insulin secretion in the pancreatic beta-cell.

Isoproterenol increases Na+-K+-ATPase activity by membrane insertion of alpha-subunits in lung alveolar cells.

Catecholamines promote lung edema clearance via beta-adrenergic-mediated stimulation of active Na+ transport across the alveolar epithelium. Because alveolar epithelial type II cell Na+-K+-ATPase contributes to vectorial Na+ flux, the present study was designed to investigate whether Na+-K+-ATPase undergoes acute changes in its catalytic activity in response to beta-adrenergic-receptor stimulation. Na+-K+-ATPase activity increased threefold in cells incubated with 1 microM isoproterenol for 15 min, which also resulted in a fourfold increase in the cellular levels of cAMP. Forskolin (10 microM) also stimulated Na+-K+-ATPase activity as well as ouabain binding. The increase in Na+-K+-ATPase activity was abolished when cells were coincubated with a cAMP-dependent protein kinase inhibitor. This stimulation, however, was not due to protein kinase-dependent phosphorylation of the Na+-K+-ATPase alpha-subunit; rather, it was the result of an increased number of alpha-subunits recruited from the late endosomes into the plasma membrane. The recruitment of alpha-subunits to the plasma membrane was prevented by stabilizing the cortical actin cytoskeleton with phallacidin or by blocking anterograde transport with brefeldin A but was unaffected by coincubation with amiloride. In conclusion, isoproterenol increases Na+-K+-ATPase activity in alveolar type II epithelial cells by recruiting alpha-subunits into the plasma membrane from an intracellular compartment in an Na+-independent manner.

Dopamine-dependent inhibition of jejunal Na+-K+-ATPase during high-salt diet in young but not in adult rats.

During high-salt diet endogenous dopamine (DA) reduces jejunal sodium transport in young but not in adult rats. This study was designed to evaluate whether this effect is mediated, at the cellular level, by inhibition of Na+-K+-ATPase activity. Enzyme activity was determined in isolated jejunal cells by the rate of [gamma-32P]ATP hydrolysis. Cells were obtained from weanling and adult rats fed either with high- or normal-salt diet. In 20-day-old but not in 40-day-old rats Na+-K+-ATPase activity was significantly reduced during high-salt diet. This inhibition was abolished by a blocker of DA synthesis. The decreased activity was associated with a decreased alpha1-subunit at the plasma membrane. During high-salt diet there was an increase in DA content in jejunal cells from 20-day-old rats, associated with a parallel decrease in 5-hydroxytryptamine, compared with normal-salt diet. In 40-day-old rats, however, the catecholamine level remained unchanged during high-salt diet. Incubation of isolated jejunal cells with DA resulted in a dose-dependent inhibition of Na+-K+-ATPase activity in 20- but not in 40-day-old rats. We conclude that during high-salt diet, jejunal Na+-K+-ATPase in 20-day-old rats is inhibited, and this effect is likely to be mediated by locally formed DA.

Protein kinase A induces recruitment of active Na+,K+-ATPase units to the plasma membrane of rat proximal convoluted tubule cells.

1. The aim of this study was to investigate the mechanism of control of Na+,K+-ATPase activity by the cAMP-protein kinase A (PKA) pathway in rat proximal convoluted tubules. For this purpose, we studied the in vitro action of exogenous cAMP (10-3 M dibutyryl-cAMP (db-cAMP) or 8-bromo-cAMP) and endogenous cAMP (direct activation of adenylyl cyclases by 10-5 M forskolin) on Na+,K+-ATPase activity and membrane trafficking. 2. PKA activation stimulated both the cation transport and hydrolytic activity of Na+,K+-ATPase by about 40%. Transport activity stimulation was specific to the PKA signalling pathway since (1) db-cAMP stimulated the ouabain-sensitive 86Rb+ uptake in a time- and dose-dependent fashion; (2) this effect was abolished by addition of H-89 or Rp-cAMPS, two structurally different PKA inhibitors; and (3) this stimulation was not affected by inhibition of protein kinase C (PKC) by GF109203X. The stimulatory effect of db-cAMP on the hydrolytic activity of Na+,K+-ATPase was accounted for by an increased maximal ATPase rate (Vmax) without alteration of the efficiency of the pump, suggesting that cAMP-PKA pathway was implicated in membrane redistribution control. 3. To test this hypothesis, we used two different approaches: (1) cell surface protein biotinylation and (2) subcellular fractionation. Both approaches confirmed that the cAMP-PKA pathway was implicated in membrane trafficking regulation. The stimulation of Na+,K+-ATPase activity by db-cAMP was associated with an increase (+40%) in Na+, K+-ATPase units expressed at the cell surface which was assessed by Western blotting after streptavidin precipitation of biotinylated cell surface proteins. Subcellular fractionation confirmed the increased expression in pump units at the cell surface which was accompanied by a decrease (-30%) in pump units located in the subcellular fraction corresponding to early endosomes. 4. In conclusion, PKA stimulates Na+,K+-ATPase activity, at least in part, by increasing the number of Na+-K+ pumps in the plasma membrane in proximal convoluted tubule cells.

Phosphatidylinositol 3-kinase-mediated endocytosis of renal Na+, K+-ATPase alpha subunit in response to dopamine.

Dopamine (DA) inhibition of Na+,K+-ATPase in proximal tubule cells is associated with increased endocytosis of its alpha and beta subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na+,K+-ATPase alpha subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na+, K+-ATPase alpha subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D1 receptor subtype and the sequential activation of phospholipase A2, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na+,K+-ATPase alpha subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.

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 alpha- and beta-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 alpha-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 alpha-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 alpha-subunit cDNA. Treatment of this cell line with dopamine resulted in phosphorylation and endocytosis of the alpha-subunit with a concomitant decrease in Na+,K+-ATPase activity. In contrast, none of these effects were observed in cells transfected with the rodent alpha-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 alpha-subunit is essential for Na+,K+-ATPase endocytosis and that both events are responsible for the decreased enzyme activity in response to dopamine.

Receptor-mediated inhibition of renal Na(+)-K(+)-ATPase is associated with endocytosis of its alpha- and beta-subunits.

The mechanisms involved in receptor-mediated inhibition of Na(+)-K(+)-ATPase remain poorly understood. In this study, we evaluate whether inhibition of proximal tubule Na(+)-K(+)-ATPase activity by dopamine is linked to its removal from the plasma membrane and internalization into defined intracellular compartments. Clathrin-coated vesicles were isolated by sucrose gradient centrifugation and negative lectin selection, and early and late endosomes were separated on a flotation gradient. Inhibition of Na(+)-K(+)-ATPase activity by dopamine, in contrast to its inhibition by ouabain, was accompanied by a sequential increase in the abundance of the alpha-subunit in clathrin-coated vesicles (1 min), early endosomes (2.5 min), and late endosomes (5 min), suggesting its stepwise translocation between these organelles. A similar pattern was found for the beta-subunit. The increased incorporation of both subunits in all compartments was blocked by calphostin C. The results demonstrate that the dopamine-induced decrease in Na(+)-K(+)-ATPase activity in proximal tubules is associated with internalization of its alpha- and beta-subunits into early and late endosomes via a clathrin-dependent pathway and that this process is protein kinase C dependent. The presence of Na(+)-K(+)-ATPase subunits in endosomes suggests that these compartments may constitute normal traffic reservoirs during pump degradation and/or synthesis.

Dopamine stimulates sodium transport and liquid clearance in rat lung epithelium.

Pulmonary edema clearance is driven primarily by active sodium transport out of the alveoli, mediated predominantly by apical sodium channels and the basolateral NA,K-ATPase. We postulated that dopamine, analogous to its effects in other transporting epithelia, could regulate these sodium transport mechanisms and affect lung liquid clearance. We therefore studied the effects of dopamine on sodium transport and liquid clearance in isolated perfused rat lungs. Instillation of dopamine into the airways caused a dose-dependent increase in liquid clearance from isolated rat lungs of up to 33% above control values at 10(-8) to 10(-4) M concentrations. 10(-6) M amiloride, which selectively inhibits apical sodium channels, decreased basal liquid clearance by 34% but did not inhibit the dopamine-mediated stimulation of lung liquid clearance. Instillation of 10(-4) M amiloride into rat airways, which inhibits other sodium transport mechanisms non-selectively, decreased basal lung liquid clearance by 49% and inhibited the dopamine-mediated stimulation of lung liquid clearance. Perfusion of rat lungs with 5 x 10(-4) M ouabain to specifically inhibit Na,K-ATPase reduced both basal clearance (by 55%) and the dopamine-stimulated increase in lung fluid clearance. Conceivably, the stimulation of lung liquid clearance by dopamine is due to a modulation of Na,K-ATPase in the pulmonary epithelium.

Differential expression of Na-K-ATPase isoforms in rat alveolar epithelial cells.

Lung Na-K-ATPase has been shown to contribute to vectorial Na+ transport and edema clearance. The alpha 1- and beta 1-Na-K-ATPase subunits have been localized to alveolar type II (ATII) cells, and the alpha 2-Na-K-ATPase has been reported in rat lung homogenates. Expression of Na-K-ATPase alpha 1-, alpha 2-, and beta 1-subunits was investigated in rat ATII cells cultured for 7 days, a period during which they lose their phenotypic markers and differentiate to an alveolar type I (ATI)-like cell phenotype. Differentiation of ATII cells to an ATI-like phenotype resulted in a decrease of alpha 1- and an increase of alpha 2-mRNA and protein abundance without changes in the beta 1-subunit. Thus ATI-like cells exhibited a mixture of alpha 1- and alpha 2-isoforms. Nuclear run-on analysis suggests that these changes were transcriptionally regulated. The existence of the distinct functional classes of Na-K-ATPase in ATII and ATI-like cells was confirmed by ouabain inhibition of Na-K-ATPase activity. Ouabain inhibition of ATII cells was consistent with expression of the alpha 1-isozyme [50% inhibitory concentration (IC50) = 4 x 10(-5) M], whereas, in ATI-like cells, it was consistent with the presence of both alpha 1- and alpha 2-isozymes (IC50 = 9.0 x 10(-5) and 1.5 x 10(-7) M, respectively); [3H]ouabain binding studies corroborated these findings. Our results indicate that, during ATII cell cytodifferentiation with time in culture, there is a shift in isoform composition that may reflect physiological functions of alveolar epithelial cells.