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.     

Regulation of Na+,K(+)-ATPase activity by dopamine in cultured rat aortic smooth muscle cells

We investigated the effect of dopamine on Na+,K(+)-ATPase activity in cultured aortic smooth muscle cells. Na+,K(+)- ATPase activity was measured by a coupled enzyme assay. Our results demonstrate that dopamine and dopamine receptor agonists, SKF-38393 (a D1 receptor agonist) and quinpirole (a D2 receptor agonist) produced 62%, 50% and 49% inhibition of Na+,K(+)-ATPase activity in aortic smooth muscle cells, respectively. The combination of the two agonists produced inhibition similar to that of dopamine. Dopamine- and the agonist-induced Na+,K(+)-ATPase inhibition was blocked by selective receptor antagonists. The Na+,K(+)-ATPase inhibition by SKF-38393 but not by quinpirole was abolished by pertussis toxin. Na+,K(+)-ATPase inhibition was also achieved by guanosine triphosphate analog GTP-gamma-S. SKF-38393 but not quinpirole stimulated phosphoinositide hydrolysis rate in rat aortic slices. SKF-38393-induced phosphoinositide hydrolysis stimulation was reversed by SCH-23390, a dopamine D1 receptor antagonist, and attenuated by pertussis toxin. In conclusion, our observations indicate that dopamine and dopamine receptor agonists inhibit Na+,K(+)-ATPase activity through specific vascular receptors. Dopamine D1 receptors are linked to pertussis toxin sensitive-mechanism(s) and a GTP-binding protein appears to be coupled to the enzyme inhibition. Finally, the inhibition of Na+,K(+)-ATPase activity in response to dopamine D1 receptor activation may be mediated by the phospholipase C signaling pathway.     

Altered arachidonic acid metabolism contributes to the failure of dopamine to inhibit Na+,K(+)-ATPase in kidney of spontaneously hypertensive rats

Dopamine decreases tubular sodium reabsorption in part by inhibition of Na+,K(+)-ATPase activity in renal proximal tubules. The signaling mechanism involved in dopamine-mediated inhibition of Na+,K(+)-ATPase is known to be defective in spontaneously hypertensive animals. The present study was designed to evaluate the role of phospholipase A2 (PLA2) and its metabolic pathway in dopamine-induced inhibition of Na+,K(+)-ATPase in renal proximal tubules from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Renal proximal tubular suspensions were prepared and Na+,K(+)-ATPase activity was measured as ouabain-sensitive adenosine triphosphate hydrolysis. Dopamine inhibited Na+,K(+)-ATPase activity in a concentration (1 nM-10 microM)-dependent manner in WKY rats while it failed to inhibit the enzyme activity in SHR. Dopamine (10 microM)-induced inhibition of Na+,K(+)-ATPase activity in WKY rats was significantly blocked by mepacrine (10 microM), a PLA2 inhibitor, suggesting the involvement of PLA2 in dopamine-mediated inhibition of Na+,K(+)-ATPase. Arachidonic acid (a product released by PLA2 action) inhibited Na+,K(+)-ATPase in a concentration-dependent (1-100 microM) manner in WKY rats while the inhibition in SHR was significantly attenuated (IC50: 7.5 and 80 microM in WKY rats and SHR, respectively). Furthermore, lower concentrations of arachidonic acid stimulated (30% at 1 microM) Na+,K(+)-ATPase activity in SHR. This suggests a defect in the metabolism of arachidonic acid in SHR. Proadifen (10 microM), an inhibitor of cytochrome P-450 monoxygenase (an arachidonic acid metabolizing enzyme) significantly blocked the inhibition produced by arachidonic acid in WKY rats and abolished the difference in arachidonic acid inhibition of Na+,K(+)-ATPase between WKY rats and SHR. These data suggest that PLA2 is involved in dopamine-induced inhibition of Na+,K(+)-ATPase and altered arachidonic acid metabolism may contribute to reduced dopaminergic inhibition of Na+,K(+)-ATPase activity in spontaneously hypertensive rats.     

Early prognostic biochemical indicators of fulminant hepatic failure

This article reviews related studies from the authors' laboratory, which focus on the regulation of vascular Na+,K+-ATPase in hypertension. Earlier studies, including the authors', suggested that Na-pump activity in cardiovascular tissues is subject to regulation during hypertension; most of these studies report a stimulation of the vascular enzyme during established stages of hypertension. To test hypothesis that in vascular smooth muscle, strain resulting from elevated pressure may be a signal initiating a cascade of events leading to increased expression of Na+,K+-ATPase, the authors used cell culture and the Flexercell Strain Unit to apply cyclical stretch to rat aortic smooth muscle cells (ASMC) for several days. These studies demonstrated that mechanical strain induces the upregulation of both the alpha-1 and alpha-2 subunits of Na+,K+-ATPase. Mechanisms underlying these changes appear to involve a transient increase in intracellular sodium entering the cell through stretch-activated channels. Calcium entering the cell via L-type channels did not affect stretch-induced upregulation of the alpha isoforms. In addition, protein kinase C inhibition resulted in inhibition of the Na-pump during stretch, but not under nonstretch conditions. The authors conclude that the stretch component of vascular pressure upregulates the Na+,K+-ATPase catalytic subunits. Intracellular sodium may be a signal for this regulation. In addition, phosphorylation by PKC may be important in stretch-induced short-term regulation of the vascular Na-pump.     

Antinociceptive effects of SC-39566, an opioid dipeptide arylalkylamide, in the Rhesus monkey

We stably expressed the rat D1A dopamine receptor in mouse fibroblast LTK- cells and obtained specific ligand binding and functional activity characteristic of the D1A dopamine receptor coupled to stimulation of adenylyl cyclase. In the transfected cells, the selective D1 agonist fenoldopam caused a concentration-dependent inhibition of Na+/K(+)-ATPase activity, achieving maximum inhibition of approximately 30%. The latter was abolished by the selective D1 antagonist (+)-SCH 23390 and by the specific protein kinase A inhibitor protein kinase inhibitor-(6-22) amide. In the nontransfected cells, fenoldopam did not affect Na+/K(+)-ATPase activity. 8-Chlorophenylthio-cAMP inhibited Na+/K(+)-ATPase activity in both transfected and nontransfected cells; this effect was blocked by protein kinase inhibitor-(6-22). These results indicate that the inhibition of Na+/K(+)-ATPase activity induced by agonist occupancy of D1A receptors is mediated by protein kinase A.     

cAMP-dependent phosphorylation of two sites in the alpha subunit of the cardiac sodium channel

The voltage-sensitive Na+ channel is responsible for generating action potentials in the heart which are critical for coordinated cardiac muscle contraction. Cardiac Na+ channels are regulated by cAMP-dependent phosphorylation, but the sites of phosphorylation are not known. Using mammalian cells expressing the rat cardiac Na+ channel (rH1) alpha subunit and site-specific antibodies, we have shown that the alpha subunit of rat heart Na+ channel is phosphorylated selectively by cAMP-dependent protein kinase (PKA) in vitro and in intact cells. Analysis of the sites of phosphorylation by two-dimensional phosphopeptide mapping and site-directed mutagenesis of fusion proteins revealed that the cardiac alpha subunit is phosphorylated selectively in vitro by PKA on Ser526 and Ser529 in the intracellular loop connecting homologous domains I and II (LI-II). These two residues were phosphorylated in intact cells expressing the rH1 alpha subunit when PKA was activated. Our results define a different pattern of phosphorylation of LI-II of cardiac and brain Na+ channels and implicate phosphorylation of Ser526 and Ser529 in the differential regulation of cardiac and brain Na+ channels by PKA.     

Axonal contact regulates expression of alpha2 and beta2 isoforms of Na+, K+-ATPase in Schwann cells: adhesion molecules and nerve regeneration

Three isoforms of catalytic alpha subunits and two isoforms of beta subunits of Na+,K+-ATPase were detected in rat sciatic nerves by western blotting. Unlike the enzyme in brain, sciatic nerve Na+,K+-ATPase was highly resistant to ouabain. The ouabain-resistant alpha1 isoform was demonstrated to be the predominant form in rat intact sciatic nerve by quantitative densitometric analysis and is mainly responsible for sciatic nerve Na+,K+-ATPase activity. After sciatic nerve injury, the alpha3 and beta1 isoforms completely disappeared from the distal segment owing to Wallerian degeneration. In contrast, alpha2 and beta2 isoform expression and Na+,K+-ATPase activity sensitive to pyrithiamine (a specific inhibitor of the alpha2 isoform) were markedly increased in Schwann cells in the distal segment of the injured sciatic nerve. These latter levels returned to baseline with nerve regeneration. Our results suggest that alpha3 and beta1 isoforms are exclusive for the axon and alpha2 and beta2 isoforms are exclusive for the Schwann cell, although axonal contact regulates alpha2 and beta2 isoform expressions. Because the beta2 isoform of Na+,K+-ATPase is known as an adhesion molecule on glia (AMOG), increased expression of AMOG/beta2 on Schwann cells in the segment distal to sciatic nerve injury suggests that AMOG/beta2 may act as an adhesion molecule in peripheral nerve regeneration.     

Stimulation of ouabain-sensitive 86Rb+ uptake and Na+,K+-ATPase alpha-subunit phosphorylation by a cAMP-dependent signalling pathway in intact cells from rat kidney cortex

We investigated in intact cortical kidney tubules the role of PKA-mediated phosphorylation in the short-term control of Na+,K+-ATPase activity. The phosphorylation level of Na+,K+-ATPase was evaluated after immunoprecipitation of the enzyme from 32P-labelled cortical tubules and the cation transport activity of Na+,K+-ATPase was measured by ouabain-sensitive 86Rb+ uptake. Incubation of cells with cAMP analogues (8-bromo-cAMP, dibutyryl-cAMP) or with forskolin plus 3-isobutyl-1-methylxanthine increased the phosphorylation level of the Na+,K+-ATPase alpha-subunit and stimulated ouabain-sensitive 86Rb+ uptake. Inhibition of PKA by H-89 blocked the effects of dibutyryl-cAMP on both phosphorylation and 86Rb+ uptake processes. The results suggest that phosphorylation by PKA stimulates the Na+,K+-ATPase activity.     

A putative fourth Na+,K(+)-ATPase alpha-subunit gene is expressed in testis

The Na+,K(+)-ATPase alpha subunit has three known isoforms, alpha 1, alpha 2 and alpha 3, each encoded by a separate gene. This study was undertaken to determine the functional status of a fourth human alpha-like gene, ATP1AL2. Partial genomic sequence analysis revealed regions exhibiting sequence similarity with exons 3-6 of the Na+,K(+)-ATPase alpha isoform genes. ATP1AL2 cDNAs spanning the coding sequence of a novel P-type ATPase alpha subunit were isolated from a rat testis library. The predicted polypeptide is 1028 amino acids long and exhibits 76-78% identity with the rat Na+,K(+)-ATPase alpha 1, alpha 2 and alpha 3 isoforms, indicating that ATP1AL2 may encode a fourth Na+,K(+)-ATPase alpha isoform. A 3.9-kb mRNA is expressed abundantly in human and rat testis.     

Na+,K(+)-ATPase phosphorylation in the choroid plexus: synergistic regulation by serotonin/protein kinase C and isoproterenol/cAMP-PK/PP-1 pathways

BACKGROUND: The ion pump Na+,K(+)-ATPase is responsible for the secretion of cerebrospinal fluid from the choroid plexus. In this tissue, the activity of Na+,K(+)-ATPase is inhibited by serotonin via stimulation of protein kinase C-catalyzed phosphorylation. The choroid plexus is highly enriched in two phosphoproteins which act as regulators of protein phosphatase-1 activity, DARPP-32 and inhibitor-1. Phosphorylation catalyzed by cAMP-dependent protein kinase on a single threonyl residue converts DARPP-32 and inhibitor-1 into potent inhibitors of protein phosphatase-1. Previous work has shown that in the choroid plexus, phosphorylation of DARPP-32 and I-1 is enhanced by isoproterenol and other agents that activate cAMP-PK. We have now examined the possible involvement of the cAMP-PK/protein phosphatase-1 pathway in the regulation of Na+,K(+)-ATPase. MATERIALS AND METHODS: The state of phosphorylation of Na+,K(+)-ATPase was measured by determining the amount of radioactivity incorporated into the ion pump following immunoprecipitation from 32P-prelabeled choroid plexuses incubated with various drugs (see below). Two-dimensional phosphopeptide mapping was employed to identify the protein kinase involved in the phosphorylation of Na+,K(+)-ATPase. RESULTS: The serotonin-mediated increase in Na+,K(+)-ATPase phosphorylation is potentiated by okadaic acid, an inhibitor of protein phosphatases-1 and -2A, as well as by forskolin or the beta-adrenergic agonist, isoproterenol, activators of cAMP-dependent protein kinase. Two-dimensional phosphopeptide maps suggest that this potentiating action occurs at the level of a protein kinase C phosphorylation site. Forskolin and isoproterenol also stimulate the phosphorylation of DARPP-32 and protein phosphatase inhibitor-1, which in their phosphorylated form are potent inhibitors of protein phosphatase-1. CONCLUSIONS: The results presented here support a model in which okadaic acid, forskolin, and isoproterenol achieve their synergistic effects with serotonin through phosphorylation of DARPP-32 and inhibitor-1, inhibition of protein phosphatase-1, and a reduction of dephosphorylation of Na+,K(+)-ATPase at a protein kinase C phosphorylation site.     

Dopamine fails to stimulate protein kinase C activity in renal proximal tubules of spontaneously hypertensive rats

We have previously reported that dopamine-1 receptor-mediated activation of phospholipase C is diminished in renal cortical slices of spontaneously hypertensive rats. The present study was carried out to examine the effect of dopamine on protein kinase C (PKC), which is one of the enzymes involved in the signal-transduction pathway leading to dopamine-induced inhibition of Na+/K(+)-ATPase in the renal proximal tubule. Renal proximal tubule suspensions were obtained from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats of 10-12 weeks old. The tubules were incubated with dopamine in the presence or absence of DA-1 receptor antagonist SCH 23390. The PKC activity was measured by using a specific fluorescent peptide substrate (sequence, PKSRTLSVAAK). We found that dopamine produced a concentration-dependent increase in protein kinase C activity in the WKY rats, however, it failed to stimulate PKC activity in the SHR. Peak stimulation of 3.828 +/- 0.35 (ng/micrograms) protein in the WKY rats was observed at dopamine concentration of 1 microM, which was blocked in a concentration-dependent manner by SCH 23390 (0.25 microM). These results provide evidence that dopamine directly stimulates PKC activity via activation of DA-1 receptors in WKY rats. Furthermore, we discovered that dopamine fails to stimulate PKC activity in the SHR. This phenomenon may be responsible for the failure of dopamine to inhibit Na+/K(+)-ATPase activity in the hypertensive animals.     

A Glu329-->Gln variant of the alpha-subunit of the rat kidney Na+,K(+)-ATPase can sustain active transport of Na+ and K+ and Na+,K(+)-activated ATP hydrolysis with normal turnover number

An allelic variant of the ouabain-insensitive rat kidney Na+,K(+)-ATPase alpha 1-isoform was identified by chance in a cDNA library. The variant differed from the wild-type rat kidney Na+,K(+)-ATPase by a single G-to-C base substitution in the cDNA, which on amino acid level gave rise to a glutamine in place of the glutamate residue Glu329 previously suggested as a likely donator of oxygen ligands for Na+ and K+ binding. The variant cDNA was transfected into COS-1 cells and the transfectants expanded with success into stable cell lines that were able to grow in the presence of a concentration of ouabain highly cytotoxic to the parental cells containing only the endogenous COS-1 cell Na+,K(+)-ATPase. Under these conditions, the viability of the cells depended on the cation transport mediated by the ouabain-insensitive Glu329-->Gln variant, whose cDNA was shown by polymerase chain reaction amplification to be stably integrated into the COS-1 cell genome. The maximum specific ATP hydrolysis activity of isolated plasma membranes of the Glu329-->Gln variant did not differ significantly from that of plasma membranes containing the wild type. A method was established for measurement of the phosphorylation capacity of the expressed Glu329-->Gln variant and wild-type enzyme, and it was thereby demonstrated that the variant had a turnover number similar if not identical to that of the wild-type.     

Glutamate uptake stimulates Na+,K+-ATPase activity in astrocytes via activation of a distinct subunit highly sensitive to ouabain

The excitatory amino acid glutamate was previously shown to stimulate aerobic glycolysis in astrocytes by a mechanism involving its uptake through an Na+-dependent transporter. Evidence had been provided that Na+,K+-ATPase might be involved in this process. We have now measured the activity of Na+,K+-ATPase in cultured astrocytes, using ouabain-sensitive 86Rb uptake as an index. L-Glutamate increases glial Na+,K+-ATPase activity in a concentration-dependent manner with an EC50 = 67 microM. Both L- and D-aspartate, but not D-glutamate, produce a similar response, an observation that is consistent with an uptake-related effect rather than a receptor-mediated one. Under basal conditions, concentration-dependent inhibition of Na+,K+-ATPase activity in astrocytes by ouabain indicates the presence of a single catalytic site with a low affinity for ouabain (K0.5 = 113 microM), compatible with the presence of an alpha1 isozyme. On stimulation with glutamate, however, most of the increased activity is inhibited by low concentrations of ouabain (K0.5 = 20 nM), thus revealing a high-affinity site akin to the alpha2 isozyme. These results suggest that astrocytes possess a glutamate-sensitive isoform of Na+,K+-ATPase that can be mobilized in response to increased neuronal activity.     

Angiotensin II AT1 receptor/signaling mechanisms in the biphasic effect of the peptide on proximal tubular Na+,K+-ATPase

The present study was designed to determine the cellular signaling mechanisms responsible for mediating the effects of angiotensin II on proximal tubular Na+,K+-ATPase activity. Angiotensin II produced a biphasic effect on Na+,K+-ATPase activity: stimulation at 10(-13) - 10(-10) M followed by inhibition at 10(-7) - 10(-5) M of angiotensin II. The stimulatory and inhibitory effects of angiotensin II were antagonized by losartan (1nM) suggesting the involvement of AT1 receptor. Angiotensin II produced inhibition of forskolin-stimulated cAMP accumulation at 10(-13) - 10(-10) M followed by a stimulation in basal cAMP levels at 10(-7) - 10(-5) M. Pretreatment of proximal tubules with losartan (1nM) antagonized both the stimulatory and inhibitory effects of angiotensin II on cAMP accumulation. Pretreatment of the proximal tubules with pertussis toxin (PTx) abolished the stimulation of Na+,K+-ATPase activity but did not affect the inhibition of Na+,K+-ATPase activity produced by angiotensin II. Pretreatment of the tubules with cholera toxin did not alter the biphasic effect of angiotensin II on Na+,K+-ATPase activity. Mepacrine (10microM), a phospholipase A2 (PLA2) inhibitor, reduced only the inhibitory effect of angiotensin II on Na+,K+-ATPase activity. These results suggest that the activation of AT1 angiotensin II receptors stimulates Na+,K+-ATPase activity via a PTx-sensitive G protein-linked inhibition of adenylyl cyclase pathway, whereas the inhibition of Na+,K+-ATPase activity following AT1 receptor activation involves multiple signaling pathways which may include stimulation of adenylyl cyclase and PLA2.     

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.     

Structure-function study on gastric proton pump

H+, K(+)-ATPase is a proton pump responsible for gastric acid secretion. It actively transport proton and K+ coupled with the hydrolysis of ATP, resulting in the formulation of a 10(6) fold proton gradient across the plasma membrane of parietal cells. The pump belongs to a family of P-type ATPases which include the Na+ pump (Na+, K(+)-ATPase) and the Ca2+ pump (Ca(2+)-ATPase). This review focuses on the structure-function relationship of this proton pump by using functional antibodies, specific inhibitor(s), a fluorescent reagent and site-directed mutants. First we prepared monoclonal antibodies which modified the functions of the H+, K(+)-ATPase . One of the antibodies, HK2032 inhibited the H+, K(+)-ATPase activity and the chloride conductance in gastric vesicles opened by S-S cross-linking, suggesting that the chloride pathway is in the H+, K(+)-ATPase molecule, and that the H+, K(+)-ATPase is a multi-functional molecule. Other antibody, HK4001 inhibited the H+, K(+)-ATPase activity by inhibiting its phosphorylation step. By using this antibody we found an H+, K(+)-ATPase isoform in the rabbit distal colon. Second we found that scopadulcic acid B, a main ingredient of Paraguayan traditional herb, is an inhibitor specific for the H+, K(+)-ATPase. This compound inhibited the H+, K(+)-ATPase activity by stabilizing the K(+)-form of the enzyme. Third we studied the conformational changes of the H+, K(+)-ATPase by observing the fluorescence of FITC-labeled enzyme. H+, K(+)-ATPase did not utilize acetylphosphate instead the ATP as an energy source of active transport, suggesting that the energy transduction system is not common among P-type ATPases. Finally we constructed a functional expression system of the H+, K(+)-ATPase in human kidney cells. By using this functional expression system in combination with site-directed mutagenesis, we studied the significance of amino acid residues in the catalytic centers (a phosphorylation site and an ATP binding site) and the putative cation binding sites. We newly found the sites determining the affinity for cations.     

Maturation of rat renal tubular response to alpha-adrenergic agonists and neuropeptide Y: a study on the regulation of Na+,K+-ATPase

Na+,K+-ATPase in tubular cells plays a pivotal role for the regulation of renal sodium excretion. In adult rats the activity of this enzyme is inhibited by natriuretic hormones and stimulated by antinatriuretic hormones. Here we have examined the tubular response to alpha-adrenergic agonists and neuropeptide Y (NPY) in both infant and adult rats. In the adult kidney, alpha-adrenergic agonists and NPY stimulate Na+,K+-ATPase activity via Ca2+-dependent pathways. Oxymetazoline, a selective alpha-adrenergic agonist, and NPY failed to stimulate proximal tubular (PT) Na+,K+-ATPase activity in 10-d-old rats in doses of 10(-8) to 10(-5) M and 10(-8) to 10(-6) M, respectively, but when tubules were incubated simultaneously with both oxymetazoline 10(-8) M and NPY 5 x 10(-9) M, stimulation was observed in both 10- and 40-d-old rat PT. This effect was abolished by FK 506, an inhibitor of Ca2+ and calmodulin-dependent protein phosphatase 2B in both age groups. A23187, a calcium ionophore, stimulated Na+,K+-ATPase in both infant and adult PT, but 10-fold higher doses were required for the infant tubules. The effect of alpha-adrenergic agonists and NPY on free intracellular Ca2+ was studied in PT cells in primary culture. The Ca2+ response to each agent was less pronounced in infant than in adult cells. Preincubation with NPY, which increases Ca2+ influx into the cells, enhanced the response to the alpha-adrenergic agonist in both infant and adult cells. The results support the concept that the systems regulating renal tubular Na+, K+-ATPase and sodium metabolism undergo postnatal maturation.     

Regulation of the Na+/K(+)-ATPase pump in vitro after long-term exposure to cocaine: role of serotonin

Long-term exposure to cocaine can cause persistent behavioral changes and alterations in neuronal function. One cocaine-regulated mRNA in the rat brain is the beta-1 subunit of the Na+/K(+)-ATPase pump. We examined both Na+/K(+)-ATPase function and expression after cocaine treatment of pheochromocytoma cells. One-hour exposure to cocaine did not alter Na+/K(+)-ATPase activity, as measured by the ouabain-sensitive component of rubidium uptake. Four days of cocaine resulted in an approximately 30% decrease in Na+/K(+)-ATPase activity. Western blot analyses demonstrated an approximately 25% decrease in levels of the beta-1 isoform, without changes in pump total alpha subunit levels. Treatment with dopamine type 1 or type 2 receptor agonists for the same period did not affect Na+/K(+)-ATPase activity. The serotonin-selective reuptake inhibitor paroxetine caused an approximately 45% decrease in rubidium uptake after 4 days, whereas pump function was not altered after treatment with either the dopamine-selective reuptake blocker nomifensine or the norepinephrine-selective reuptake blocker desipramine. Chronic treatment with both cocaine and LY 278,584, a serotonin type 3 receptor antagonist, did not replicate the cocaine-associated decrease in pump function. Long-term cocaine exposure regulates expression and function of the Na+/K(+)-ATPase pump in neuronal-like cells; this regulation is mediated in part via the serotonin type 3 receptor. Similar Na+/K(+)-ATPase pump regulation in vivo may selectively alter neuronal function in the mammalian brain.     

Endothelin 1 stimulates Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport through ETA receptors and protein kinase C-dependent pathway in cerebral capillary endothelium

The effect of endothelins (ET-1 and ET-3) on 86Rb+ uptake as a measure of K+ uptake was investigated in cultured rat brain capillary endothelium. ET-1 or ET-3 dose-dependently enhanced K+ uptake (EC50 = 0.60 +/- 0.15 and 21.5 +/- 4.1 nM, respectively), which was inhibited by the selective ETA receptor antagonist BQ 123 (cyclo-D-Trp-D-Asp-Pro-D-Val-Leu). Neither the selective ETB agonists IRL 1620 [N-succinyl-(Glu9,-Ala11,15)-ET-1] and sarafotoxin S6c, nor the ETB receptor antagonist IRL 1038 [(Cys11,Cys15)-ET-1] had any effect on K+ uptake. Ouabain (inhibitor of Na+,K(+)-ATPase) and bumetanide (inhibitor of Na(+)-K(+)-Cl- cotransport) reduced (up to 40% and up to 70%, respectively) the ET-1-stimulated K+ uptake. Complete inhibition was seen with both agents. Phorbol 12-myristate 13-acetate (PMA), activator of protein kinase C (PKC), stimulated Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport. ET-1- but not PMA-stimulated K+ uptake was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (inhibitor of Na+/H+ exchange system), suggesting a linkage of Na+/H+ exchange with ET-1-stimulated Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport activity that is not mediated by PKC.