PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic beta cells.

Hypoglycemic sulfonylureas represent a group of clinically useful antidiabetic compounds that stimulate insulin secretion from pancreatic beta cells. The molecular mechanisms involved are not fully understood but are believed to involve inhibition of potassium channels sensitive to adenosine triphosphate (KATP channels) in the beta cell membrane, causing membrane depolarization, calcium influx, and activation of the secretory machinery. In addition to these effects, sulfonylureas also promoted exocytosis by direct interaction with the secretory machinery not involving closure of the plasma membrane KATP channels. This effect was dependent on protein kinase C (PKC) and was observed at therapeutic concentrations of sulfonylureas, which suggests that it contributes to their hypoglycemic action in diabetics.

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.

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.

Dopamine regulation of renal Na+,K(+)-ATPase activity is lacking in Dahl salt-sensitive rats.

Dopamine is a natriuretic hormone that acts by inhibiting tubular Na+, K(+)-ATPase activity by activation of the dopamine-1 receptor (the thick ascending limb [TAL] of Henle) or by a synergistic effect of dopamine-1 and dopamine-2 receptors (the proximal tubule). The dopamine-1 receptor is coupled to adenylate cyclase. In this article we show that prehypertensive Dahl salt-sensitive (DS) rats have a blunted natriuretic response to dopamine determined during euvolemic conditions compared with Dahl salt-resistant (DR) rats. Furthermore, we have examined the renal tubular effects of dopamine in DS and DR rats. Basal Na+,K(+)-ATPase activity was similar in DS and DR rats. In proximal tubule, dopamine (10(-5) M) inhibited Na+,K(+)-ATPase activity in DR but not in DS rats. The dopamine-2 agonist LY171555 (10(-5) M) together with dibutyryl cyclic AMP (10(-6) M) inhibited proximal tubule Na+,K(+)-ATPase activity in both DS and DR rats. LY171555 alone had no effect. In TAL, the dopamine-1 agonist fenoldopam (10(-5) M) inhibited Na+,K(+)-ATPase activity in DR but not in DS rats. Dibutyryl cyclic AMP (10(-5) M) inhibited TAL Na+,K(+)-ATPase activity in both DS and DR rats. In cell suspensions from the cortex and the medulla, activation of the dopamine-1 receptor significantly increased cyclic AMP content in DR but not in DS rats. The results indicate that DS rats lack the capacity to inhibit tubular Na+,K(+)-ATPase activity because of a defective dopamine-1 receptor adenylate cyclase coupling. This defect may contribute to the impaired natriuretic capacity in DS rats.

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.

Antagonistic actions of renal dopamine and 5-hydroxytryptamine: increase in Na+, K(+)-ATPase activity in renal proximal tubules via activation of 5-HT1A receptors.

1. 5-Hydroxytryptamine (5-HT) is antinatriuretic. Since this effect of 5-HT is not accomplished by changes in glomerular haemodynamics, we have examined in this study whether 5-HT may influence sodium excretion by affecting the Na+, K(+)-ATPase activity in renal cortical tubules. 2. Na+, K(+)-ATPase activity was determined as the rate of [32P]-ATP hydrolysis in renal cortical tubules in suspension. Basal Na+, K(+)-ATPase activity in renal tubules was 4.8 +/- 0.4 mumol Pi mg-1 protein h-1 (n = 8). The 5-HT1A receptor agonist, (+/-)-8-hydroxy-2-(di-n-propylamino) tetraline (8-OH-DPAT) (10 to 3000 nM) induced a concentration-dependent increase (P < 0.05) in Na+, K(+)-ATPase activity with an EC50 value of 355 nM (95% confidence limits: 178, 708). Maximal stimulation elicited by 3000 nM of 8-OH-DPAT was antagonized by the selective 5-HT1A receptor antagonist, (+)-WAY 100135 10 to 1000 nM) with an IC50 value of 20 nM (14, 29); 0.3 microM (+)-WAY 100135 completely abolished (P < 0.01) the stimulatory effect of 8-OH-DPAT. The stimulatory effect of 8-OH-DPAT was found to be time-dependent (15 +/- 2% and 66 +/- 7% increase at 2.5 and 5.0 min, respectively). The 5-HT2 receptor agonist alpha-methyl-5-HT (100 to 3000 nM) did not induce any significant changes in Na+, K(+)-ATPase activity (5.0 +/- 1.5 mumol Pi mg-1 protein h-1; n = 4). 3. The stimulatory effect 8-OH-DPAT was absent when homogenates were used. Stimulation occurred at a Vmax concentration (70 mM) of sodium supporting the notion that stimulation occurs independently of increasing sodium permeability. 4. The inhibitory effect of dopamine (P < 0.05) on Na+, K(+)-ATPase activity was blunted by co-incubation with 8-OH-DPAT (0.5 microM). 5. It is concluded that activation of 5-HT1A receptors increases Na+, K(+)-ATPase activity in renal cortical tubules; this effect may represent an important cellular mechanism, at the tubule level, responsible for the antinatriuretic effect of 5-HT.

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.

Studies on the nature of the antagonistic actions of dopamine and 5-hydroxytryptamine in renal tissues.

The present work examines the possibility of whether the reciprocal effects of dopamine (DA) and 5-hydroxytryptamine (5-HT) are only dependent on the antagonistic nature of the signal resulting from the activation of their specific receptors or may also result from a competitive type of inhibition at different levels of the synthetic and metabolic pathways shared by DA and 5-HT. Studies performed in isolated proximal convoluted tubules (PCT) have shown that L-5-HTP and L-DOPA use the same transporter in order to be taken up into the cell and both L-DOPA and L-5-HTP exert a competitive type of inhibition upon their cellular uptake. The decrease in the formation of 5-HT in isolated PCT induced by L-DOPA reflects most probably a reduction in the intracellular availability of L-5-HTP. However, in experiments conducted in homogenates of PCT L-DOPA was found to be a better substrate for AAAD than L-5-HTP. Apart from sharing a common synthetic pathway, DA and 5-HT also share a common metabolic pathway; type A monoamine oxidase (MAO-A), the predominant form of MAO in rat renal tissues, converts DA into 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-HT into 5-hydroxyindolacetic acid (5-HIAA). However, in contrast to 5-HT, DA can be metabolized by MAO-B and catechol-O-methyltransferase. Inhibition of MAO-A was found to produce a 2-fold increase in the urinary excretion of 5-HT; this increase in the urinary excretion of 5-HT was accompanied by an unexpected reduction in the urinary excretion of DA.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Diacylglycerol activation of protein kinase C results in a dual effect on Na+,K(+)-ATPase activity from intact renal proximal tubule cells.

This study evaluated the effect of L-1-oleoyl-2-acetyl-sn-3-glycerol (OAG) on ouabain-sensitive Na,K-dependent oxygen consumption (Na,K-QO2) in intact renal proximal tubule cells (RPTC). Basal Na,K-QO2 (nmol O2/mg protein per min) was 20.0 +/- 1.0. Incubation with 10 nM of OAG induced a dual effect on Na,K-QO2, with an initial stimulation (maximal at 10 min, 37.1 +/- 5.0), followed by an inhibition (significant at 20 min, 16.3 +/- 1.0). No changes in ouabain-insensitive QO2 were observed in any of the protocols. The effects were abolished by sphingosine, a protein kinase C inhibitor. Stimulation was abolished by amiloride 0.1 mM. Amiloride had no effect on Na,K-QO2 at the concentration used. Stimulation was not potentiated by the sodium ionophore, amphotericin B, and the later inhibition was still observed in the presence of amphotericin B. The initial stimulation was attributed to an increase in sodium permeability, while the later inhibition was attributed to a direct effect on the Na,K-pump. Regulation of Na+,K(+)-ATPase activity by protein kinase C in intact RPTC can be accomplished by a direct effect on the protein or as a secondary effect consequent upon changes in intracellular sodium.

Short-term regulation of renal Na-K-ATPase activity: physiological relevance and cellular mechanisms.

Sodium-potassium-activated adenosinetriphosphatase (Na-K-ATPase; the Na:K pump), located at the basolateral domain of epithelial cells, provides the driving force for active sodium and potassium translocation and for the secondary active transport of other solutes across the renal tubules. Short-term regulation of renal Na-K-ATPase activity (i.e., not reflecting changes in its biosynthesis rate) provides an important mechanism of modulating tubule transport and thus the final Na and K urinary excretion. Recent studies have provided abundant evidence that such regulation is effected by complex functional networks that are specific for different nephron segments and involve distinct and often mutually interacting intracellular signal transduction pathways. The effects of hormones and autacoids linked to alterations in cell adenosine 3',5'-cyclic monophosphate and consequently of protein kinase A, in the levels and distribution of protein kinase C, or in the generation of various eicosanoids provide examples of rapid Na:K pump activity modulation by the mechanisms mentioned above. In this review we assess the roles of specific intracellular messengers and the manner in which they, and especially protein kinases, might interact with the pump in the short-term regulation of its activity; also, we examine the emerging evidence supporting the participation of the cytoskeleton in this process.

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.

Renal Na+,K(+)-ATPase in Dahl salt-sensitive rats: K+ dependence, effect of cell environment and protein kinases.

Na+,K(+)-ATPase in renal epithelial cells plays an important role in the regulation of Na+ balance, extracellular volume and blood pressure. The function of renal Na+,K(+)-ATPase in Dahl salt-sensitive (DS) rats, an animal model for salt-sensitive hypertension, and Dahl salt-resistant (DR) rats has been studied. In Na+,K(+)-ATPase partially purified from renal cortex, affinities and the Hill coefficients for Na+ and K+ activation were similar in DS and DR rats. Only one component of low ouabain affinity site was found in both strains, indicating the presence of the alpha 1 isoform. Protein kinase C and cAMP-dependent protein kinase phosphorylated Na+,K(+)-ATPase alpha subunit in DS and DR rats, and the phosphorylation by protein kinase C was associated with an inhibition of enzyme activity. The kinetic parameters for K+ activation were also studied in a preparation of basolateral membranes and were found to be similar in DS and DR rats. In a preparation of cortical tubule cells, Na+,K(+)-ATPase activity was determined as ouabain-sensitive oxygen consumption (OS QO2). Maximal OS QO2, measured in Na+ loaded cells, was the same in DS and DR rats. The K0.5 for K+ was significantly lower in DS than DR rats (0.163 +/- 0.042 vs. 0.447 +/- 0.061 mM, P < 0.05), indicating that factors regulating Na+,K(+)-ATPase activity in intact cells are altered in DS rats. Kinetic parameters for Na+ activation in cells were the same in both strains. In summary, the function of renal Na+,K(+)-ATPase molecule is not altered in DS rats.(ABSTRACT TRUNCATED AT 250 WORDS)

High salt diet down-regulates proximal tubule Na+, K(+)-ATPase activity in Dahl salt-resistant but not in Dahl salt-sensitive rats: evidence of defective dopamine regulation.

We examined the regulation of Na+,K(+)-ATPase activity in proximal tubule segments during a high salt diet in prehypertensive Dahl salt-sensitive and salt-resistant rats. Rats were placed on normal salt or high salt diets (0.9% saline as drinking water). During the normal salt diet, Na+,K(+)-ATPase activity was not different between Dahl salt-sensitive and salt-resistant rats. After 2 days and 10 days on a high salt diet, Na+,K(+)-ATPase activity in Dahl salt-resistant rats significantly decreased when compared to Dahl salt-resistant rats on a normal salt diet (P less than 0.01). The decreased Na+,K(+)-ATPase activity in Dahl salt-resistant rats during a high salt diet was reversed by treatment with an inhibitor of aromatic L-amino acid decarboxylase (dopamine synthesizing enzyme), benserazide. In contrast, Na+,K(+)-ATPase activity did not decrease during the high salt diet and benserazide had no effect on Na+,K(+)-ATPase activity in Dahl salt-sensitive rats. These results indicate that Dahl salt-sensitive rats do not have the capacity to down-regulate the proximal tubule Na+,K(+)-ATPase activity during a high salt diet. Indirect evidence suggests that the regulation of Na+,K(+)-ATPase activity by locally produced dopamine is absent in Dahl salt-sensitive rats.

Endogenous dopamine modulates jejunal sodium absorption during high-salt diet in young but not in adult rats.

BACKGROUND/AIMS: This study was designed to investigate the contribution of endogenous catecholamines to the regulation of small intestinal sodium transport during postnatal development. METHODS: Jejunal permeability was determined by a constant perfusion, nonabsorbable marker technique in weanling, adolescent, and adult rats fed either a high-salt diet or normal-salt diet. Tissue catecholamine levels were determined by high-performance liquid chromatography with electrochemical detection. RESULTS: In 20-day-old but not in 40-day-old rats, a significantly lower net sodium absorption was observed during high-salt diet compared with age-matched controls on normal-salt diet. Inhibition of dopamine synthesis significantly increased the net sodium absorption in 20-day-old rats on high-salt diet compared with untreated 20-day-old rats on high-salt diet. The basal levels of dopamine in 20-day-old rats were twofold higher than in 40-day-old rats. During high-salt diet, both age groups responded with an increase in dopamine production. Norepinephrine levels were significantly higher (30-fold) in 20-day-old rats than in 40-day-old rats, but norepinephrine content was not significantly changed during high-salt diet in either groups. CONCLUSIONS: The results indicate that weanling animals have a greater jejunal sodium absorption than older animals, probably because of higher noradrenergic tonus. A challenge with a high-salt diet results in a decrease of the intestinal sodium absorption in weaning rats but not in adult rats; endogenous dopamine appears to play an important role in this regulation.

Activation of epithelial Na+ channels by protein kinase A requires actin filaments.

We have recently demonstrated a novel role for "short" actin filaments, a distinct species of polymerized actin different from either monomeric (G-actin) or long actin filaments (F-actin), in the activation of epithelial Na+ channels. In the present study, the role of actin in the activation of apical Na+ channels by the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A (PKA) was investigated by patch-clamp techniques in A6 epithelial cells. In excised inside-out patches, addition of deoxyribonuclease I, which prevents actin polymerization, inhibited Na+ channel activation mediated by PKA. Disruption of endogenous actin filament organization with cytochalasin D for at least 1 h prevented the PKA-mediated activation of Na+ channels but not activation following the addition of actin to the cytosolic side of the patch. To assess the role of PKA on actin filament organization, actin was used as a substrate for the specific phosphorylation by the PKA. Actin was phosphorylated by PKA with an equilibrium stoichiometry of 2:1 mol PO4-actin monomer. Actin was phosphorylated in its monomeric form, but only poorly once polymerized. Furthermore, phosphorylated actin reduced the rate of actin polymerization. Thus actin allowed to polymerize for at least 1 h in the presence of PKA and ATP to obtain phosphorylated actin filaments induced Na+ channel activity in excised inside-out patches, in contrast to actin polymerized either in the absence of PKA or in the presence of PKA plus a PKA inhibitor (nonphosphorylated actin filaments). This was also confirmed by using purified phosphorylated G-actin incubated in a polymerizing buffer for at least 1 h at 37 degrees C. These data suggest that the form of actin required for Na+ channel activation (i.e., "short" actin filaments) may be favored by the phosphorylation of G-actin and may thus mediate or facilitate the activation of Na+ channels by PKA.

Dissociation between changes in cytoplasmic free Ca2+ concentration and insulin secretion as evidenced from measurements in mouse single pancreatic islets.

Simultaneous measurements of cytosolic free Ca2+ concentration and insulin release, in mouse single pancreatic islets, revealed a direct correlation only initially after stimulation with glucose or K+. Later, there is an apparent dissociation between these two parameters, with translocation of alpha and epsilon isoenzymes of protein kinase C to membranes and simultaneous desensitization of insulin release in response to glucose. Recovery of insulin release, without any concomitant changes in cytosolic free Ca2+ concentration, after addition of phorbol 12-myristate 13-acetate, okadaic acid, and forskolin supports the notion that the desensitization process is accounted for by dephosphorylation of key regulatory sites of the insulin exocytotic machinery.

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.

Phosphorylation of the catalytic subunit of Na+,K(+)-ATPase inhibits the activity of the enzyme.

We have examined two distinct protein kinases, cAMP-dependent protein kinase and protein kinase C, for their ability to phosphorylate and regulate the activity of three different types of Na+,K(+)-ATPase preparation. cAMP-dependent protein kinase phosphorylated purified shark rectal gland Na+,K(+)-ATPase to a stoichiometry of approximately 1 mol of phosphate per mol of alpha subunit. Protein kinase C phosphorylated purified shark rectal gland Na+,K(+)-ATPase to a stoichiometry of approximately 2 mol of phosphate per mol of alpha subunit. The phosphorylation by each of the kinases was associated with an inhibition of Na+,K(+)-ATPase activity of about 40-50%. These two protein kinases also inhibited the activity of a partially purified preparation of Na+,K(+)-ATPase from rat renal cortex and the activity of Na+,K(+)-ATPase present in preparations of basolateral membrane vesicles from rat renal cortex.