Ouabain-sensitive ATP Hydrolysis Research Articles

Src Family Kinase Links Insulin Signaling to Short Term Regulation of Na,K-ATPase in Nonpigmented Ciliary Epithelium.

Insulin has been shown to elicit changes of Na,K-ATPase activity in various tissues. Na,K-ATPase in the nonpigmented ciliary epithelium (NPE) plays a role in aqueous humor secretion and changes of Na,K-ATPase activity impact the driving force. Because we detect a change of NPE Na,K-ATPase activity in response to insulin, studies were carried out to examine the response mechanism. Ouabain-sensitive rubidium (Rb) uptake by cultured NPE cells, measured as a functional index of Na,K-ATPase-mediated inward potassium transport, was found to increase in cells exposed for 5 min to insulin. The maximally effective concentration was 100 nM. An intrinsic increase of Na,K-ATPase activity evident as a >2-fold increase in the rate of ouabain-sensitive ATP hydrolysis in homogenates obtained from cells exposed to 100 nM insulin for 5 min was also observed. Insulin-treated cells exhibited Akt, Src family kinase (SFK), ERK1/2, and p38 activation, all of which were prevented by a pI3 kinase inhibitor LY294002. The Rb uptake and Na,K-ATPase activity response to insulin both were abolished by PP2, an SFK inhibitor which also prevented p38 and ERK1/2 but not Akt activation. The Akt inhibitor MK-2206 did not change the Na,K-ATPase response to insulin. The findings suggest insulin activates pI3K-dependent Akt and SFK signaling pathways that are separate. ERK1/2 and p38 activation is secondary to and dependent on SFK activation. The increase of Na,K-ATPase activity is dependent on activation of the SFK pathway. The findings are consistent with previous studies that indicate a link between Na,K-ATPase activity and SFK signaling. J. Cell. Physiol. 232: 1489-1500, 2017. © 2016 Wiley Periodicals, Inc.

Uroguanylin modulates (Na+ + K+)ATPase in a proximal tubule cell line: Interactions among the cGMP/protein kinase G, cAMP/protein kinase A, and mTOR pathways

BackgroundThe natriuretic effect of uroguanylin (UGN) involves reduction of proximal tubule (PT) sodium reabsorption. However, the target sodium transporters as well as the molecular mechanisms involved in these processes remain poorly understood. MethodsTo address the effects of UGN on PT (Na++K+)ATPase and the signal transduction pathways involved in this effect, we used LLC-PK1 cells. The effects of UGN were determined through ouabain-sensitive ATP hydrolysis and immunoblotting assays during different experimental conditions. ResultsWe observed that UGN triggers cGMP/PKG and cAMP/PKA pathways in a sequential way. The activation of PKA leads to the inhibition of mTORC2 activity, PKB phosphorylation at S473, PKB activity and, consequently, a decrease in the mTORC1/S6K pathway. The final effects are decreased expression of the α1 subunit of (Na++K+)ATPase and inhibition of enzyme activity. ConclusionsThese results suggest that the molecular mechanism of action of UGN on sodium reabsorption in PT cells is more complex than previously thought. We propose that PKG-dependent activation of PKA leads to the inhibition of the mTORC2/PKB/mTORC1/S6K pathway, an important signaling pathway involved in the maintenance of the PT sodium pump expression and activity. General significanceThe current results expand our understanding of the signal transduction pathways involved in the overall effect of UGN on renal sodium excretion.

TRPV4 Channels Enable Na,K‐ATPase Activity in the Lens Epithelium to Respond to a Remote Stimulus

Na,K-ATPase activity is low or absent in the fiber cells that make up most of the eye lens. Ion and water homeostasis of the entire structure is made possible by Na,K-ATPase activity in a single epithelial cell layer at the anterior lens surface. Previously we have shown Src family tyrosine kinase-dependent changes of Na,K-ATPase activity in the epithelium. Here we provide evidence that the epithelium is able to increase Na,K-ATPase activity in response to a remote stimulus in the fiber mass. The posterior pole of intact pig lenses was subjected to localized damage either by a 2.5 mm diameter freeze-thaw wound or by injection of 5 µl of hyperosmotic saline just beneath the surface. After 2-30 min the epithelium was removed from the lens, homogenized and the rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was determined. Src family tyrosine kinase (SFK) activation (phosphorylation) was assessed by Western blot. Localized damage to lens fibers at posterior pole caused SFK activation and a significant increase in Na,K-ATPase activity in the epithelium that occurred within minutes. Na,K-ATPase activity almost doubled. The increase in Na,K-ATPase activity was prevented by the SFK inhibitor PP2 (10 µM). Importantly, the SFK phosphorylation response as well as the increase in Na,K-ATPase activity were suppressed by a TRPV4 channel antagonist, HC067947 (10 µM). By Western blot, TRPV4 was detected in the epithelium but not fiber cells. The findings point to a TRPV4-dependent mechanism in the anterior epithelium that enables the cells to sense remote damage in the fiber mass and respond by activating SFK and increasing Na,K-ATPase activity.

Nitric Oxide Regulation of Na, K‐ATPase Activity in Ocular Ciliary Epithelium Involves Src Family Kinase

The nitric oxide (NO) donor sodium nitroprusside (SNP) is known to reduce aqueous humor (AH) secretion in the isolated porcine eye. Previously, SNP was found to inhibit Na,K-ATPase activity in nonpigmented ciliary epithelium (NPE), AH-secreting cells, through a cGMP/protein kinase G (PKG)-mediated pathway. Here we show Src family kinase (SFK) activation in the Na,K-ATPase activity response to SNP. Ouabain-sensitive (86) Rb uptake was reduced by >35% in cultured NPE cells exposed to SNP (100 µM) or exogenously added cGMP (8-Br-cGMP) (100 µM) and the SFK inhibitor PP2 (10 µM) prevented the response. Ouabain-sensitive ATP hydrolysis was reduced by ~40% in samples detected in material obtained from SNP- and 8-Br-cGMP-treated cells following homogenization, pointing to an intrinsic change of Na,K-ATPase activity. Tyrosine-10 phosphorylation of Na,K-ATPase α1 subunit was detected in SNP and L-arginine-treated cells and the response prevented by PP2. SNP elicited an increase in cell cGMP. Cells exposed to 8-Br-cGMP displayed SFK activation (phosphorylation) and inhibition of both ouabain-sensitive (86) Rb uptake and Na,K-ATPase activity that was prevented by PP2. SFK activation, which also occurred in SNP-treated cells, was suppressed by inhibitors of soluble guanylate cyclase (ODQ; 10 µM) and PKG (KT5823; 1 µM). SNP and 8-Br-cGMP also increased phosphorylation of ERK1/2 and p38 MAPK and the response prevented by PP2. However, U0126 did not prevent SNP or 8-Br-cGMP-induced inhibition of Na,K-ATPase activity. Taken together, the results suggest that NO activates guanylate cyclase to cause a rise in cGMP and subsequent PKG-dependent SFK activation. Inhibition of Na,K-ATPase activity depends on SFK activation.

Ouabain and Digoxin Preconditioning Protect Cardiac Na/K‐ATPase against Ischemia‐Reperfusion Injury in Langendorff‐perfused Mouse Hearts

Protection against myocardial Ischemia‐Reperfusion (IR) injury by Ischemic PreConditioning (IPC) involves the protection of Na/K‐ATPase activity. The cardiac glycosides (CG) digoxin and ouabain are specific inhibitors of Na/K‐ATPase‐mediated ion transport, but also trigger preconditioning through activation of Na/K‐ATPase signaling. In Langendorff‐perfused mouse hearts, we investigated whether CG preconditioning also protects Na/K‐ATPase.Forty min of ischemia followed by 30 min of reperfusion decreased the left ventricular developed pressure (LVDP) by 71.4% (60.8 ± 2.3 vs 17.4 ± 1.9 mmHg, P<0.01), induced a lactate dehydrogenase (LDH) release of 5.8 ±1.2 U, and resulted in an infarct of 52.9 ± 5.4% of the area at risk (as measured by tissue staining). Na/K‐ATPase enzyme activity measured by ouabain‐sensitive ATP hydrolysis was decreased by 59% (3.2 ± 0.89 vs 7.8 ± 0.6 umol/h/mg, P<0.05) without change in Na/K‐ATPase α or β subunit content as shown by Western blotting. In hearts exposed to 10 μM ouabain (OPC) or digoxin (DigPC), or 4 cycles of brief ischemia (IPC) prior to prolonged ischemia, LVDP recovery was nearly doubled (P<0.01 vs IR), infarct size was reduced by half (P<0.01 vs IR ), and LDH released was decreased by 70%(P<0.05 vs IR).These suggest that, like IPC, OPC and its more clinically relevant counterpart DigPC protect cardiac Na/K‐ATPase activity during IR. (NIH P01HL036573)

Prolactin and dopamine 1-like receptor interaction in renal proximal tubular cells

Prolactin is a natriuretic hormone and acts by inhibiting the activity of renal tubular Na(+)-K(+)-ATPase activity. These effects require an intact renal dopamine system. Here, we have studied by which mechanism prolactin and dopamine interact in Sprague-Dawley rat renal tissue. Na(+)-K(+)-ATPase activity was measured as ouabain-sensitive ATP hydrolysis in microdissected renal proximal tubular segments. Intracellular signaling pathways were studied by a variety of different techniques, including Western blotting using phosphospecific antibodies, immunoprecipitation, and biotinylation assays. We found that dopamine and prolactin regulated Na(+)-K(+)-ATPase activity via similar signaling pathways, including protein kinase A, protein kinase C, and phosphoinositide 3-kinase activation. The cross talk between prolactin and dopamine 1-like receptors was explained by a heterologous recruitment of dopamine 1-like receptors to the plasma membrane in renal proximal tubular cells. Prolactin had no effect on Na(+)-K(+)-ATPase activity in spontaneously hypertensive rats, a rat strain with a blunted response to dopamine. These results further emphasize the central role of the renal dopamine system in the interactive regulation of renal tubular salt balance.

Prostaglandin E2 modulates Na+,K+‐ATPase activity in rat hippocampus: implications for neurological diseases

Prostaglandin E(2) (PGE(2)) is quantitatively one of the major prostaglandins synthesized in mammalian brain, and there is evidence that it facilitates seizures and neuronal death. However, little is known about the molecular mechanisms involved in such excitatory effects. Na(+),K(+)-ATPase is a membrane protein which plays a key role in electrolyte homeostasis maintenance and, therefore, regulates neuronal excitability. In this study, we tested the hypothesis that PGE(2) decreases Na(+),K(+)-ATPase activity, in order to shed some light on the mechanisms underlying the excitatory action of PGE(2). Na(+),K(+)-ATPase activity was determined by assessing ouabain-sensitive ATP hydrolysis. We found that incubation of adult rat hippocampal slices with PGE(2) (0.1-10 microM) for 30 min decreased Na(+),K(+)-ATPase activity in a concentration-dependent manner. However, PGE(2) did not alter Na(+),K(+)-ATPase activity if added to hippocampal homogenates. The inhibitory effect of PGE(2) on Na(+),K(+)-ATPase activity was not related to a decrease in the total or plasma membrane immunocontent of the catalytic alpha subunit of Na(+),K(+)-ATPase. We found that the inhibitory effect of PGE(2) (1 microM) on Na(+),K(+)-ATPase activity was receptor-mediated, as incubation with selective antagonists for EP1 (SC-19220, 10 microM), EP3 (L-826266, 1 microM) or EP4 (L-161982, 1 microM) receptors prevented the PGE(2)-induced decrease of Na(+),K(+)-ATPase activity. On the other hand, incubation with the selective EP2 agonist (butaprost, 0.1-10 microM) increased enzyme activity per se in a concentration-dependent manner, but did not prevent the inhibitory effect of PGE(2). Incubation with a protein kinase A (PKA) inhibitor (H-89, 1 microM) and a protein kinase C (PKC) inhibitor (GF-109203X, 300 nM) also prevented PGE(2)-induced decrease of Na(+),K(+)-ATPase activity. Accordingly, PGE(2) increased phosphorylation of Ser943 at the alpha subunit, a critical residue for regulation of enzyme activity. Importantly, we also found that PGE(2) decreases Na(+),K(+)-ATPase activity in vivo. The results presented here imply Na(+),K(+)-ATPase as a target for PGE(2)-mediated signaling, which may underlie PGE(2)-induced increase of brain excitability.

Inhibition of Na-K-ATPase in thick ascending limbs by NO depends on O2- and is diminished by a high-salt diet.

A high-salt diet enhances nitric oxide (NO)-induced inhibition of transport in the thick ascending limb (THAL). Long exposures to NO inhibit Na-K-ATPase in cultured cells. We hypothesized that NO inhibits THAL Na-K-ATPase after long exposures and a high-salt diet would augment this effect. Rats drank either tap water or 1% NaCl for 7-10 days. Na-K-ATPase activity was assessed by measuring ouabain-sensitive ATP hydrolysis by THAL suspensions. After 2 h, spermine NONOate (SPM; 5 microM) reduced Na-K-ATPase activity from 0.44 +/- 0.03 to 0.30 +/- 0.04 nmol P(i).microg protein(-1).min(-1) in THALs from rats on a normal diet (P < 0.03). Nitroglycerin also reduced Na-K-ATPase activity (P < 0.04). After 20 min, SPM had no effect (change -0.07 +/- 0.05 nmol P(i).microg protein(-1).min(-1)). When rats were fed high salt, SPM did not inhibit Na-K-ATPase after 120 min. To investigate whether ONOO(-) formed by NO reacting with O(2)(-) was involved, we measured O(2)(-) production. THALs from rats on normal and high salt produced 35.8 +/- 0.3 and 23.7 +/- 0.8 nmol O(2)(-).min(-1).mg protein(-1), respectively (P < 0.01). Because O(2)(-) production differed, we studied the effects of the O(2)(-) scavenger tempol. In the presence of 50 microM tempol, SPM did not inhibit Na-K-ATPase after 120 min (0.50 +/- 0.05 vs. 0.52 +/- 0.07 nmol P(i).microg protein(-1).min(-1)). Propyl gallate, another O(2)(-) scavenger, also prevented SPM-induced inhibition of Na-K-ATPase activity. SPM inhibited pump activity in tubules from rats on high salt when O(2)(-) levels were increased with xanthine oxidase and hypoxanthine. We concluded that NO inhibits Na-K-ATPase after long exposures when rats are on a normal diet and this inhibition depends on O(2)(-). NO donors do not inhibit Na-K-ATPase in THALs from rats on high salt due to decreased O(2)(-) production.

Clathrin-mediated Endocytosis of Na+,K+-ATPase in Response to Parathyroid Hormone Requires ERK-dependent Phosphorylation of Ser-11 within the α1-Subunit

Parathyroid hormone (PTH) inhibits Na(+),K(+)-ATPase activity through protein kinase C- (PKC) and extracellular signal-regulated kinase- (ERK) dependent pathways and increases serine phosphorylation of the alpha(1)-subunit. To determine whether specific serine phosphorylation sites within the Na(+),K(+)-ATPase alpha(1)-subunit are involved in the Na(+),K(+)-ATPase responses to PTH, we examined the effect of PTH in opossum kidney cells stably transfected with wild type rat Na(+),K(+)-ATPase alpha(1)-subunit (WT), serine 11 to alanine mutant alpha(1)-subunit (S11A), or serine 18 to alanine mutant alpha(1)-subunit (S18A). PTH increased phosphorylation and endocytosis of the Na(+),K(+)-ATPase alpha(1)-subunit into clathrin-coated vesicles in cells transfected with WT and S18A rat Na(+),K(+)-ATPase alpha(1)-subunits. PTH did not increase the level of phosphorylation or stimulate translocation of Na(+),K(+)-ATPase alpha(1)-subunits into clathrin-coated vesicles in cells transfected with the S11A mutant. PTH inhibited ouabain-sensitive (86)Rb uptake and Na(+),K(+)-ATPase activity (ouabain-sensitive ATP hydrolysis) in WT- and S18A-transfected opossum kidney cells but not in S11A-transfected cells. Pretreatment of the cells with the PKC inhibitors and ERK inhibitor blocked PTH inhibition of (86)Rb uptake, Na(+),K(+)-ATPase activity, alpha(1)-subunit phosphorylation, and endocytosis in WT and S18A cells. Consistent with the notion that ERK phosphorylates Na(+),K(+)-ATPase alpha(1)-subunit, ERK was shown to be capable of causing phosphorylation of Na(+),K(+)-ATPase alpha(1)-subunit immunoprecipitated from WT and S18A but not from S11A-transfected cells. These results suggest that PTH regulates Na(+),K(+)-ATPase by PKC and ERK-dependent alpha(1)-subunit phosphorylation and that the phosphorylation requires the expression of a serine at the 11 position of the Na(+),K(+)-ATPase alpha(1)-subunit.

D-glucose stimulates the Na+/K+ pump in mouse pancreatic islet cells.

To determine the effect of D-glucose on the β-cell Na+/K+ pump, 86Rb+ influx was studied in isolated, -cell-rich islets of Umeå-ob/ob mice in the absence or presence of lmM ouabain. D-glucose (20 mM) stimulated the ouabain-sensitive portion of 86Rb+ influx by 65%, whereas the ouabain-resistant portion was inhibited by 48%. The Na+/K+ ATPase activity in homogenates of islets of Umeå-ob/ob mice or normal mice was determined to search for direct effects of D-glucose. Thus, ouabain-sensitive ATP hydrolysis in islet homogenates was measured in the presence of different D-glucose concentrations. No effect of D-glucose (3–20 mM) was observed in either ob/ob or normal islets at the optimal Na+/K+ ratio for the enzyme (135 mM Na+ and 20 mM K+). Neither D-glucose (3–20 mM) nor L-glucose or 3-O-methyl-D-glucose (20 mM) affected the enzyme activity at a high Na+/K+ ratio (175 mM Na+ and 0.7mM K+). Diphenylhydantoin (150 μM) decreased the enzyme activity at optimal Na+/K+ ratio, whereas 50 μM of the drug had no effect. The results suggest that D-glucose induces a net stimulation the Na+/K+ pump of β-cells in intact islets and that D-glucose does not exert any direct effect on the Na+/K+ ATPase activity.

Mechanism of FK 506/520 action on rat renal proximal tubular Na+,K+-ATPase activity

The neurotransmitter in renal sympathetic nerves, norepinephrine (NE), regulates the activity of proximal tubule (PT) Na+,K+-ATPase in a bidirectional manner via stimulation of alpha- and beta-adrenoceptors. The stimulatory alpha-adrenergic pathway is mediated by calcineurin, the target molecule for FK 506 and related compounds. We examined whether the FK 506 analogue FK 520, by interrupting the calcineurin-mediated alpha-adrenergic signaling pathway, enhance the inhibitory beta-adrenergic effect of NE on PT Na+,K+-ATPase activity. The effects of three days of treatment with FK 520 were examined on rat renal PT Na+,K+-ATPase activity, measured as ouabain-sensitive ATP hydrolysis in single, microdissected PT segments. Renal function studies, including glomerular filtration rate (GFR) and urinary excretion of N-acetyl-3-D-glucoseaminidase (NAG), were examined using conventional clearance techniques after three days of treatment with FK 506. FK 520 treatment induced a pronounced and dose-dependent decrease in PT Na+,K+-ATPase activity. This effect was completely reversed by the competitive FK 520 antagonist, L 685 818, indicating that the effect was dependent on inhibition of calcineurin. To test whether the FK 520-induced decrease in Na+, K+-ATPase activity was mediated by enhanced beta-adrenoceptor signaling, the FK 520 effect was examined in rats pretreated with a beta-adrenoceptor antagonist (propranolol) or rats subjected to renal denervation. Both of these procedures prevented the FK 520-induced decrease in Na+,K+-ATPase activity. Thus, during FK 520 treatment, renal sympathetic nerves mediate an inhibitory effect on PT Na+,K+-ATPase activity via beta-adrenoceptors. Propranolol pretreatment also prevented FK 506-induced decreases in GFR and urinary excretion of NAG, an index of PT dysfunction. The results support the hypothesis that the net effect of the neurotransmitter NE on Na+,K+-ATPase activity is dependent on the balance between the alpha- and beta-adrenergic signaling pathways and suggest that agents that interfere with these pathways may, by altering the activity of tubular Na+,K+-ATPase, also alter the function of the renal tubular epithelial cell.

Partial inhibition of Na,K-ATPase activity in cultured rabbit non-pigmented ciliary epithelium following an episode of cytoplasmic ATP depletion.

Ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was measured in digitonin-permeabilized monolayers of cultured cells derived from rabbit non-pigmented ciliary epithelium. Diminished Na,K-ATPase activity was observed in cells that had been pre-treated 10 min with the protein kinase C activator, PDBu, as well as in cells that had been cooled to 4 degrees C for 4 h then rewarmed to 37 degrees C for 30 min (cool-rewarm manoeuvre). In the intact cells, ouabain binding was not decreased either by PDBu treatment or the cool-rewarm manoeuvre. However, both PDBu and the cool-rewarm manoeuvre increased the rate of ouabain-sensitive potassium (86Rb) uptake measured in intact cells. Cell ATP content was diminished in PDBu-treated cells and cells subjected to the cool-rewarm manoeuvre. We suggest that an episode of ATP depletion might initiate a mechanism which causes lasting, partial inhibition of Na,K-ATPase activity. In keeping with this suggestion, diminished Na,K-ATPase activity was observed in cells that had been pre-treated 20 min with the metabolic inhibitors CCCP or rotenone and in cells pre-treated 2.5 h in dextrose-free medium. This study illustrates that Na,K-ATPase activity measured in the permeabilized cell is a complex parameter which is not necessarily a reliable indicator of sodium pump responses in the intact cell.

Regulation of Na+,K+-ATPase in submandibular glands of hypophysectomized male mice by steroid and thyroid hormones.

The effects of thyroid hormone, androgen, glucocorticoid, and mineralocorticoid on Na+,K+-ATPase activity and on levels of its alpha-subunit protein (alpha 1 isoform) in mouse submandibular gland (SMG) were studied by enzyme assay for ouabain-sensitive ATP hydrolysis, by quantitative densitometric scanning of Western blots, and by immunohistochemistry. To define the specific regulatory effects of various pituitary-dependent hormones on expression of Na+,K+-ATPase in the SMG, we treated hypophysectomized (hypox) male mice with triiodo-L-thyronine (T3), 5 alpha-dihydrotestosterone (DHT), dexamethasone (Dex), and aldosterone (Ald), injected singly or in combination. Na+,K+-ATPase was confined to the duct system of the SMG. In intact mice there was a gender difference in SMG Na+,K+-ATPase, with levels of the enzyme's activity and of its alpha 1-subunit being less in the glands of males. In males, hypophysectomy caused a rise in levels of Na+,K+-ATPase activity and in levels of the alpha 1-subunit protein of this enzyme, and in intensity of immunocytochemical staining for this subunit but there were no such changes in the SMG of hypox females. Changes caused by hormonal replacement to hypox males in Na+,K-ATPase activity, levels of its alpha 1-subunit, or the intensity of immunocytochemical staining for this subunit were complex. Ald had no effect. T3 or dexamethasone, given alone, induced Na+,K+-ATPase activity above control values (hypox males) and increased levels of its alpha 1-subunit protein and immunohistochemical staining for this subunit. By contrast, DHT did not cause a decline in any of these parameters. However, when treatment with T3 was combined with administration of Dex or DHT, enzymatic activity of Na+,K+-ATPase decreased but levels of the alpha 1-subunit protein and immunohistochemical staining for this subunit increased. Therefore, inductions of the alpha 1-subunit of this enzyme are not always correlated with increases in levels of activity of Na+,K+-ATPase, and we propose that both enzymatic and immunochemical analyses are essential for evaluation of hormonal regulation of Na+,K+-ATPase in salivary gland and in other tissues.

Actin filaments stimulate the Na(+)-K(+)-ATPase.

In this report, the functional role of actin on Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity was explored. The Na(+)- and K(+)-dependent, ouabain-sensitive ATP hydrolysis mediated by rat kidney Na(+)-K(+)-ATPase increased by 74% in the presence of previously unpolymerized actin (24 microM), whereas addition of polymerized actin was without effect. Addition of actin was associated instead with an increase in the affinity of the Na(+)-K(+)-ATPase for Na+ but not other enzymatic substates. A maximal stimulatory effect (296%) was observed either at an Na(+)-K(+)-ATPase:actin ratio of 1:50,000 or at lower ratios (1:625) by shifting from the E2 (K+ selective) to the E1 (Na+ selective) conformation of the enzyme. Immunoblotting of actin to the purified Na(+)-K(+)-ATPase suggested that this interaction may be linked to binding of actin to the enzyme, which was further supported by sequence analysis indicating putative actin-binding domains in the alpha-subunit of the enzyme. The interaction between actin and the Na(+)-K(+)-ATPase may imply a novel functional role of the cytoskeleton in the control of ion transport.

The influence of thapsigargin on Na,K-ATPase activity in cultured nonpigmented ciliary epithelial cells.

Experiments were conducted to test the influence of thapsigargin on the NaK-ATPase activity of cultured cells (ODM2) derived from human nonpigmented ciliary epithelium. The rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was diminished in cells that had been pretreated with thapsigargin then permeabilized. Following 20 min exposure of intact cells to thapsigargin, the cells were permeabilized with digitonin and the rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was measured immediately in a calcium-free buffer. In permeabilized cells that had been pretreated with 1 microM thapsigargin for 20 min, the rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was reduced by 38%. Pretreatment with lesser concentrations of thapsigargin caused smaller changes of Na,K-ATPase activity. The decrease of Na,K-ATPase activity was the same whether or not calmodulin antagonists W7 or trifluoperazine were present during the thapsigargin pretreatment period. This inhibitory effect upon the Na,K-ATPase may serve to limit the extent of sodium pump activation that takes place in intact cells when thapsigargin causes sodium pump stimulation by a mechanism that appears to involve changes in cytoplasmic ion levels when potassium channels open.

Surface and Intracellular Pools of Na,K-ATPase Catalytic and Immuno-activities in Rat Exorbital Lacrimal Gland

The subcellular distribution of Na,K-ATPase in rat lacrimal gland acinar cells was surveyed by subcellar fractionation followed by determination of two Na,K-ATPase catalytic activities, K+-dependent p -nitrophenylphosphate hydrolysis and ouabain-sensitive ATP hydrolysis, by Western blotting of isolated membrane fractions, and by analysis of lacrimal tissue with immunocytochemical methods at the light and EM levels. Both catalytic activities distributed in parallel after centrifugation on sorbitol gradients. They were associated with membrane samples that appeared to be derived from: (1) acinar cell basal-lateral membranes; (2) the Golgi complex: and (3) endocytic compartments. β1-subunit immunoreactivity closely paralleled catalytic activity. The α1-subunit immunoreactivity distribution suggested the presence of α1-subunits in αβ complexes and excess α1 subunits which were not assembled with β subunits and not catalytically active. In the putative basal-lateral membrane and endocytic samples, α1-reactivity was associated primarily with a 100-kDa band, while in the Golgi samples it was associated primarily with 40 and 60-kDa bands. β1-reactivity was also heterogeneous, with reactivity in basal-lateral membrane and putative endocytic samples associated with a broad band of 50-54 kDa, and reactivity in Golgi samples associated with discrete bands of 50, 52, and 54 kDa. Staining with anti-holoenzyme and anti-α1-subunit antibodies yielded strong indirect immunofluorescence signals both in plasma membrane and in intracellular regions of acinar cells, β1-like immunoreactivity was concentrated in cytoplasmic regions of acinar cells. Immunogold electron microscopy revealed positive staining with anti-holoenzyme in Golgi membranes of acinar cells and in basal-lateral membranes of duct cells. These data support the hypothesis that lacrimal acinar cells contain substantial cytoplasmic pools of Na,K-ATPase and that there is a location-dependent heterogeneity which is not detected by immunocytochemical methods.

Control of electrolyte transport in the kidney through a dopamine- and cAMP-regulated phosphoprotein, DARPP-32.

1. DARPP-32 is a phosphoprotein regulated by dopamine and cAMP. In its phosphorylated form it acts as an inhibitor of protein phosphatase-1, thereby regulating the phosphorylation state of phosphoproteins in the basal ganglia. 2. In the kidney, DARPP-32 has been detected in the medullary thick ascending limb of Henle (mTAL) and, to a lesser degree, in the proximal convoluted tubule by means of immunohistochemistry and in situ hybridization. 3. In single microdissected tubules of rat kidney, Na+, K(+)-ATPase activity, measured as ouabain-sensitive ATP hydrolysis, has been shown to be inhibited to the same degree by the DA1 agonist fenoldopam, cAMP and a synthesized and phosphorylated DARPP-32 peptide, D32(8-38). 4. It is concluded that the DA1 receptor-mediated inhibition of Na+ transport in the mTAL by dopamine occurs via cAMP accumulation and the phosphoprotein, DARPP-32.

Amiloride and amiloride analogs inhibit Na +/K +-transporting ATPase and Na +-coupled alanine transport in rat hepatocytes

Amiloride, a commonly used inhibitor of Na +-H + exchange, has been shown to exhibit a variety of nonspecific effects. Recently, the more potent amiloride analogs, 5-(N,N- dimethyl)amiloride hydrochloride (DMA) and 5-(N- ethyl-N- isopropyl)amiloride (EIA), have been used to control for the nonspecific effects of the parent compound. In the present study, we have explored the effects of these analogs on Na +/K +-transporting ATPase (Na +/K +-ATPase) and Na +-coupled alanine transport in primary rat hepatocyte cultures and rat liver plasma membranes, and we have compared the effects of these analogs with the effects of amiloride and ouabain. Amiloride, DMA, and EIA increased staedy-state Na + content and inhibited ouabain-sensitive 86Rb + uptake in a reversible, concentration-dependent, ouabain-like manner, with estimated 50% inhibitory concentrations (IC 50) of 3.0 · 10 −3 M, 5.2 · 10 −4 M, and 1.2 · 10 −4 M, respectively. Amiloride, DMA and EIA also inhibited ouabain-sensitive ATP hydrolysis in rat liver plasma membranes with similar potency (IC 50 values of 2.2 · 10 −3 M, 2.2 · 10 −3 M, and 1.7 · 10 −4 M, respectively). In separate experiments, amiloride (5 · 10 −3 M), DMA (10 −3 M), and EIA (2.5 · 10 −4 M) decreased the uptake into hepatocytes of alanine by 20%, 61%, and 59%, respectively, and further studies with DMA (10 −3 M) demonstrated that this inhibition was largely due to a decrease in the Na +-dependent fraction of alanine uptake. These findings indicate that amiloride, DMA, and EIA inhibit hepatic Na +/K +-ATPase directly, reversibly, and with a relative rank order potency of EIA > DMA > amiloride . All three compounds also inhibit the hepatic uptake of alanine, and presumably could indirectly inhibit other Na +-coupled transport processes as well.

In situ assembly states of (Na+,K+)-pump ATPase in human erythrocytes. Radiation target size analyses.

The in situ assembly state of the (Na+,K+)-pump ATPase of human erythrocytes was studied by applying the classical target theory to radiation inactivation data of the ouabain-sensitive sodium efflux and ATP hydrolysis. Erythrocytes and their extensively washed white ghosts were irradiated at -45 to -50 degrees C with an increasing dose of 1.5-MeV electron beam, and after thawing, the Na+-pump flux and/or enzyme activities were assayed. Each activity measured was reduced as a simple exponential function of radiation dose, from which a radiation sensitive mass (target size) was calculated. When intact cells were used, the target sizes for the pump and for the ATPase activities were equal and approximately 620,000 daltons. The target size for the ATPase activity was reduced to approximately 320,000 daltons if the cells were pretreated with digitoxigenin. When ghosts were used, the target size for the ATPase activity was again approximately 320,000 daltons. Our target size measurements together with other information available in literature suggest that (Na+,K+)-pump ATPase may exist in human erythrocytes either as a tetramer of alpha beta or as a dimer of alpha beta in tight association with other protein mass, probably certain glycolytic enzymes, and that this tetrameric or heterocomplex association is dissociable by digitoxigenin treatment or by extensive wash during ghost preparation.

Formation of Oriented Membrane Multilayers of Na/K‐ATPasea

The isolated membrane-bound enzyme retains its ouabain-sensitive ATP hydrolysis activity, and produces ATP-dependent Na/sup +/ and K/sup +/ fluxes when incorporated into phospholipid vesicles. The ultimate goal of this work is to determine its low resolution structure using both X-ray and neutron diffraction. A number of methods were used to impart lamellar stacking order to highly purified pig Na/K-ATPase membranes. Upon partial dehydration, x-ray diffraction from Na/K-ATPase membrane multilayers at 98% relative humidity yielded discrete reflections of 118 A periodicity, diffracting to 1/14.8 A/sup -1/, additionally, continuous diffraction to 1/10 A/sup -1/ was obtained. Subjecting the membrane multilayers to high magnetic fields improved the quality of the lamellar diffraction dramatically. Neutron diffraction studies of the partially dehydrated Na/K-ATPase membrane multilayers detected a mosaic spread of 2/sup 0/ when the samples were subjected to a magnetic field of 5 Tesla perpendicular to the membrane surface; the reflections were narrower than the camera line width; hence, the lattice disorder has also decreased significantly, although only four orders were measured.