Time course of active and passive liquid and solute movement in the isolated perfused rat lung model

The isolated perfused rat lung model (IL)is used to study alveolar epithelial transport properties. Most of theprevious studies have been done over a short period of time and havenot used the same preparation as a control and intervention group. Weevaluated whether the IL preparation could be used for a prolonged period of time (5 h) and studied the rates of activeNa⁺ transport, lung liquidclearance, and passive movement of solutes. ActiveNa⁺ transport and lung liquidclearance were stable from 1 to 5 h. The passive movement of smallsolutes (Na⁺, mannitol) did notchange significantly, and albumin movement increased slightly at thefifth hour. Total RNA isolated from IL after 5 h was intact, and theNa⁺-K⁺-ATPaseactivity in alveolar type II cells isolated at the end of 5-hexperiments was equal toNa⁺-K⁺-ATPasefunction from freshly isolated alveolar type II cells. Finally, wemeasured the stimulatory effect of the -adrenergic-agonist terbutaline and the inhibitory effect of theNa⁺-K⁺-ATPase-antagonistouabain by using the same animal as a control. Accordingly, theisolated perfused lung model is functionally stable for at least 5 h,and it could be utilized to evaluate the effect of differentinterventions by using the same preparation.

     

Salt-inducible kinase 1 is present in lung alveolar epithelial cells and regulates active sodium transport

Salt-inducible kinase 1 (SIK1) in epithelial cells mediates the increases in active sodium transport (Na⁺, K⁺-ATPase-mediated) in response to elevations in the intracellular concentration of sodium. In lung alveolar epithelial cells increases in active sodium transport in response to β-adrenergic stimulation increases pulmonary edema clearance. Therefore, we sought to determine whether SIK1 is present in lung epithelial cells and to examine whether isoproterenol-dependent stimulation of Na⁺, K⁺-ATPase is mediated via SIK1 activity. All three SIK isoforms were present in airway epithelial cells, and in alveolar epithelial cells type 1 and type 2 from rat and mouse lungs, as well as from human and mouse cell lines representative of lung alveolar epithelium. In mouse lung epithelial cells, SIK1 associated with the Na⁺, K⁺-ATPase α-subunit, and isoproterenol increased SIK1 activity. Isoproterenol increased Na⁺, K⁺-ATPase activity and the incorporation of Na⁺, K⁺-ATPase molecules at the plasma membrane. Furthermore, those effects were abolished in cells depleted of SIK1 using shRNA, or in cells overexpressing a SIK1 kinase-deficient mutant. These results provide evidence that SIK1 is present in lung epithelial cells and that its function is relevant for the action of isoproterenol during regulation of active sodium transport. As such, SIK1 may constitute an important target for drug discovery aimed at improving the clearance of pulmonary edema.     

SGK1 activates Na+-K+-ATPase in amphibian renal epithelial cells

Serum- and glucocorticoid-induced kinase 1 (SGK1) is thought to be an important regulator of Na⁺ reabsorption in the kidney. It has been proposed that SGK1 mediates the effects of aldosterone on transepithelial Na⁺ transport. Previous studies have shown that SGK1 increases Na⁺ transport and epithelial Na⁺ channel (ENaC) activity in the apical membrane of renal epithelial cells. SGK1 has also been implicated in the modulation of Na⁺-K⁺-ATPase activity, the transporter responsible for basolateral Na⁺ efflux, although this observation has not been confirmed in renal epithelial cells. We examined Na⁺-K⁺-ATPase function in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible promoter. The results showed that expression of a constitutively active mutant of SGK1 (SGK1^T_S425D) increased the transport activity of Na⁺-K⁺-ATPase 2.5-fold. The increase in activity was a direct consequence of activation of the pump itself. The onset of Na⁺-K⁺-ATPase activation was observed between 6 and 24 h after induction of SGK1 expression, a delay that is significantly longer than that required for activation of ENaC in the same cell line (1 h). SGK1 and aldosterone stimulated the Na⁺ pump synergistically, indicating that the pathways mediated by these molecules operate independently. This observation was confirmed by demonstrating that aldosterone, but not SGK1^T_S425D, induced an 2.5-fold increase in total protein and plasma membrane Na⁺-K⁺-ATPase ₁-subunit abundance. We conclude that aldosterone increases the abundance of Na⁺-K⁺-ATPase, whereas SGK1 may activate existing pumps in the membrane in response to chronic or slowly acting stimuli. sodium transport; serum- and glucocorticoid-induced kinase; A6 cells; sodium pump     

Changes in Na+-K+-ATPase activity influence cell attachment to fibronectin

Most vital cellular functions aredependent on a fine-tuned regulation of intracellular ion homeostasis.Here we have demonstrated, using COS cells that were untransfected ortransfected with wild-type rat ouabain-resistantNa⁺-K⁺-ATPase, that partial inhibition ofNa⁺-K⁺-ATPase has a dramatic influence oncell attachment to fibronectin. Ouabain dose-dependently decreasedattachment in untransfected cells and in cells expressing wild-typeNa⁺-K⁺-ATPase, but not in cells expressingouabain-insensitive Na⁺-K⁺-ATPase, whereasinhibition of Na⁺-K⁺-ATPase by loweringextracellular K⁺ concentration decreased attachment in allthree cell types. Thirty percent inhibition ofNa⁺-K⁺-ATPase significantly attenuatedattachment. Na⁺-K⁺-ATPase inhibition caused asustained increase in the intracellular Ca²⁺ concentrationthat obscured Ca²⁺ transients observed in untreated cellsduring attachment. Inhibitors of Ca²⁺ transporterssignificantly decreased attachment, but inhibition ofNa⁺/H⁺ exchanger did not. Ouabain reduced focaladhesion kinase autophosphorylation but had no effect on cell surfaceintegrin expression. These results suggest that the level ofNa⁺-K⁺-ATPase activity strongly influences cellattachment, possibly by an effect on intracellular Ca²⁺.

     

Ouabain potentiates the activation of ERK1/2 by carbachol in parotid gland epithelial cells; inhibition of ERK1/2 reduces Na(+)-K(+)-ATPase activity

The Na⁺-K⁺-ATPase and the ERK1/2 pathway appear to be linked in some fashion in a variety of cells. The Na⁺-K⁺-ATPase inhibitor ouabain can promote ERK1/2 activation. This activation involves Src, intracellular Ca²⁺ concentration ([Ca²⁺]_i) elevation, reactive oxygen species (ROS) generation, and EGF receptor (EGFR) transactivation. In contrast, ERK1/2 can mediate changes in Na⁺-K⁺-ATPase activity and/or expression. Thus signaling between ERK1/2 and Na⁺-K⁺-ATPase can occur from either direction. Whether such bidirectionality can occur within the same cell has not been reported. In the present study, we have demonstrated that while ouabain (1 mM) produces only a small (50%) increase in ERK1/2 phosphorylation in freshly isolated rat salivary (parotid acinar) epithelial cells, it potentiates the phosphorylation of ERK1/2 by submaximal concentrations of carbachol, a muscarinic receptor ligand that initiates fluid secretion. Although ERK1/2 is only modestly phosphorylated when cells are exposed to 1 mM ouabain or 10^–6 M carbachol, the combination of these agents promotes ERK1/2 phosphorylation to near-maximal levels achieved by a log order carbachol concentration. These effects of ouabain are distinct from Na⁺-K⁺-ATPase inhibition by lowering extracellular K⁺, which promotes a rapid and large increase in ERK1/2 phosphorylation. ERK1/2 potentiation by ouabain (EC₅₀ 100 µM) involves PKC, Src, and alterations in [Ca²⁺]_i but not ROS generation or EGFR transactivation. In addition, inhibition of ERK1/2 reduces Na⁺-K⁺-ATPase activity (measured as stimulation of QO₂ by carbachol and the cationophore nystatin). These results suggest that ERK1/2 and Na⁺-K⁺-ATPase may signal to each other in each direction under defined conditions in a single cell type. protein kinase C; intracellular Ca²⁺ concentration; muscarinic receptor; ₁-subunit; potassium removal     

Insulin increases the turnover rate of Na+-K+-ATPase in human fibroblasts

Insulin stimulates K⁺ transport by theNa⁺-K⁺-ATPase in human fibroblasts. In othercell systems, this action represents an automatic response to increasedintracellular [Na⁺] or results from translocation oftransporters from an intracellular site to the plasma membrane. Here weevaluate whether these mechanisms are operative in human fibroblasts.Human fibroblasts expressed the ₁ but not the₂ and ₃ isoforms ofNa⁺-K⁺-ATPase. Insulin increased the influx ofRb⁺, used to trace K⁺ entry, but did not modifythe total intracellular content of K⁺, Rb⁺, andNa⁺ over a 3-h incubation period. Ouabain increasedintracellular Na⁺ more rapidly in cells incubated withinsulin, but this increase followed insulin stimulation ofRb⁺ transport. Bumetanide did not prevent the increasedNa⁺ influx or stimulation ofNa⁺-K⁺-ATPase. Stimulation of theNa⁺-K⁺- ATPase by insulin did not produce anymeasurable change in membrane potential. Insulin did not affect theaffinity of the pump toward internal Na⁺ or the number ofmembrane-bound Na⁺-K⁺-ATPases, as assessed byouabain binding. By contrast, insulin slightly increased the affinityof Na⁺-K⁺-ATPase toward ouabain. Phorbol estersdid not mimic insulin action on Na⁺-K⁺-ATPaseand inhibited, rather than stimulated, Rb⁺ transport. Theseresults indicate that insulin increases the turnover rate ofNa⁺-K⁺-ATPases of human fibroblasts withoutaffecting their number on the plasma membrane or modifying theirdependence on intracellular [Na⁺].

     

Purification of active Na+-K+-ATPase using a new ouabain-affinity column

Ouabain, aspecific inhibitor ofNa⁺-K⁺-ATPase,was coupled to epoxy agarose via a 13-atom spacer to make an affinitycolumn that specifically bindsNa⁺-K⁺-ATPase.Na⁺-K⁺-ATPasefrom rat and dog kidney was bound to the column and was eluted as afunction of enzyme conformation, altered by adding specificcombinations of ligands.Na⁺-K⁺-ATPasefrom both sources bound to the column in the presence of Na + ATP + Mgand in solutions containing 30 mM K. No binding was observed in thepresence of Na or Na + ATP. These experiments suggest thatNa⁺-K⁺-ATPasebinds to the column under the same conditions that it binds tountethered ouabain.Na⁺-K⁺-ATPasealready bound to the column was competitively eluted with excess freeNa + ouabain or with Na + ATP. The latter eluted active enzyme. Forcomparable amounts of boundNa⁺-K⁺-ATPase,Na + ouabain and Na + ATP eluted more rat than dogNa⁺-K⁺-ATPase,consistent with the lower affinity of the ratNa⁺-K⁺-ATPasefor ouabain. The ouabain-affinity column was used to purify activeNa⁺-K⁺-ATPasefrom rat kidney microsomes and rat adrenal glomerulosa cells. Thespecific activity of the kidney enzyme was increased from ~2 to 15 µmolP_i · mg¹ · min¹.Na⁺-K⁺-ATPasepurified from glomerulosa cells that were prelabeled with [³²P]orthophosphatewas phosphorylated on the -subunit, suggesting that these cellscontain a kinase that phosphorylatesNa⁺-K⁺-ATPase.

     

Moderate-to-severe ischemic conditions increase activity and phosphorylation of the cerebral microvascular endothelial cell Na+-K+-Cl- cotransporter

Brain edema that forms during the early stages of stroke involves increased transport of Na⁺ and Cl^– across an intact blood-brain barrier (BBB). Our previous studies have shown that a luminal BBB Na⁺-K⁺-Cl^– cotransporter is stimulated by conditions present during ischemia and that inhibition of the cotransporter by intravenous bumetanide greatly reduces edema formation in the rat middle cerebral artery occlusion model of stroke. The present study focused on investigating the effects of hypoxia, which develops rapidly in the brain during ischemia, on the activity and expression of the BBB Na⁺-K⁺-Cl^– cotransporter, as well as on Na⁺-K⁺-ATPase activity, cell ATP content, and intracellular volume. Cerebral microvascular endothelial cells (CMECs) were assessed for Na⁺-K⁺-Cl^– cotransporter and Na⁺-K⁺-ATPase activities as bumetanide-sensitive and ouabain-sensitive ⁸⁶Rb influxes, respectively. ATP content was assessed by luciferase assay and intracellular volume by [³H]-3-O-methyl-D-glucose and [¹⁴C]-sucrose equilibration. We found that 30-min exposure of CMECs to hypoxia ranging from 7.5% to 0.5% O₂ (vs. 19% normoxic O₂) significantly increased cotransporter activity as did 7.5% or 2% O₂ for up to 2 h. This was not associated with reduction in Na⁺-K⁺-ATPase activity or ATP content. CMEC intracellular volume increased only after 4 to 5 h of hypoxia. Furthermore, glucose and pyruvate deprivation increased cotransporter activity under both normoxic and hypoxic conditions. Finally, we found that hypoxia increased phosphorylation but not abundance of the cotransporter protein. These findings support the hypothesis that hypoxia stimulation of the BBB Na⁺-K⁺-Cl^– cotransporter contributes to ischemia-induced brain edema formation. edema; blood-brain barrier; bumetanide; cell volume     

(Na+-K+)-stimulated ATPase in Leaves of C4 Plants: Possible Involvement in Active Transport of C4 Acids

Leaves of three C₄ plants, Setaria italica, Pennisetum typhoides,and Amaranthus paniculatus possessed five- to ten-fold higheractivities of a (Na⁺-K⁺)-dependent ATPase than those of twoC₃ plants, Oryza sativa and Rumex vesicarius. Na⁺-K⁺ ATPasefrom leaves of Amarathus exhibited an optimal pH of 7?5 andan optimal temperature of 35 ?C. It required 40 mM K⁺ and 80mM Na⁺ for maximal activity. Ouabain partially inhibited (Na⁺-K⁺)-dependentATPase activity in leaves of C₄ plants. Ouabain also blockedthe movement of label from initially formed C₄ acids into endproducts in leaves of only C₄ plants, Setaria and Amaranthusbut not in a C₃ plant, Rumex. We propose that Na⁺-K⁺ ATPasemay mediate transfer of energy during active transport of C₄acids from mesophyll into the bundle sheath.     

Na+-K+ pumps in the transverse tubular system of skeletal muscle fibers preferentially use ATP from glycolysis

The Na⁺-K⁺ pumps in the transverse tubular (T) system of a muscle fiber play a vital role keeping K⁺ concentration in the T-system sufficiently low during activity to prevent chronic depolarization and consequent loss of excitability. These Na⁺-K⁺ pumps are located in the triad junction, the key transduction zone controlling excitation-contraction (EC) coupling, a region rich in glycolytic enzymes and likely having high localized ATP usage and limited substrate diffusion. This study examined whether Na⁺-K⁺ pump function is dependent on ATP derived via the glycolytic pathway locally within the triad region. Single fibers from rat fast-twitch muscle were mechanically skinned, sealing off the T-system but retaining normal EC coupling. Intracellular composition was set by the bathing solution and action potentials (APs) triggered in the T-system, eliciting intracellular Ca²⁺ release and twitch and tetanic force responses. Conditions were selected such that increased Na⁺-K⁺ pump function could be detected from the consequent increase in T-system polarization and resultant faster rate of AP repriming. Na⁺-K⁺ pump function was not adequately supported by maintaining cytoplasmic ATP concentration at its normal resting level (8 mM), even with 10 or 40 mM creatine phosphate present. Addition of as little as 1 mM phospho(enol)pyruvate resulted in a marked increase in Na⁺-K⁺ pump function, supported by endogenous pyruvate kinase bound within the triad. These results demonstrate that the triad junction is a highly restricted microenvironment, where glycolytic resynthesis of ATP is critical to meet the high demand of the Na⁺-K⁺ pump and maintain muscle excitability. muscle fatigue; sodium-potassium-adenosinetriphosphatase; excitation-contraction coupling; T-system; excitability     

Alloxan-induced diabetes reduces sarcolemmal Na+-K+ pump function in rabbit ventricular myocytes

The effect of diabetes on sarcolemmal Na⁺-K⁺ pump function is important for our understanding of heart disease associated with diabetes and design of its treatment. We induced diabetes characterized by hyperglycemia but no other major metabolic disturbances in rabbits. Ventricular myocytes isolated from diabetic rabbits and controls were voltage clamped and internally perfused with the whole cell patch-clamp technique. Electrogenic Na⁺-K⁺ pump current (I_p, arising from the 3:2 Na⁺-to-K⁺ exchange ratio) was identified as the shift in holding current induced by Na⁺-K⁺ pump blockade with 100 µmol/l ouabain in most experiments. There was no effect of diabetes on I_p recorded when myocytes were perfused with pipette solutions containing 80 mmol/l Na⁺ to nearly saturate intracellular Na⁺-K⁺ pump sites. However, diabetes was associated with a significant decrease in I_p measured when pipette solutions contained 10 mmol/l Na⁺. The decrease was independent of membrane voltage but dependent on the intracellular concentration of K⁺. There was no effect of diabetes on the sensitivity of I_p to extracellular K⁺. Pump inhibition was abolished by restoration of euglycemia or by in vivo angiotensin II receptor blockade with losartan. We conclude that diabetes induces sarcolemmal Na⁺-K⁺ pump inhibition that can be reversed with pharmacological intervention. sodium transport; insulin; angiotensin II; cardiomyopathy; hyperglycemia     

Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells

Cardiotonic glycosides, like ouabain, inhibit Na⁺-K⁺-ATPase. Recent evidence suggests that low molar concentrations of ouabain alter cell growth. Studies were conducted to examine the effect of ouabain on Akt phosphorylation and rate of cell proliferation in opossum kidney (OK) proximal tubule cells. Cells exposed to 10 nM ouabain displayed increased Akt Ser⁴⁷³ phosphorylation, as evidenced by an increase in phospho-Akt Ser⁴⁷³ band density. Ouabain-stimulated Akt Ser⁴⁷³ phosphorylation was inhibited by pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 and wortmannin), a PLC inhibitor (edelfosine), and an Akt inhibitor. Moreover, ouabain-mediated Akt Ser⁴⁷³ phosphorylation was suppressed by reduction of extracellular calcium (EGTA) or when intracellular calcium was buffered by BAPTA-AM. An inhibitor of calcium store release (TMB-8) and an inhibitor of calcium entry via store-operated calcium channels (SKF96365) also suppressed ouabain-mediated Akt Ser⁴⁷³ phosphorylation. In fura-2 AM-loaded cells, 10 nM ouabain increased capacitative calcium entry (CCE). Ouabain at 10 nM did not significantly alter baseline cytoplasmic calcium concentration in control cells. However, treatment with 10 nM ouabain caused a significantly higher ATP-mediated calcium store release. After 24 h, 10 nM ouabain increased the rate of cell proliferation. The Akt inhibitor, BAPTA-AM, SKF96365, and cyclopiazonic acid suppressed the increase in the rate of cell proliferation caused by 10 nM ouabain. Ouabain at 10 nM caused a detectable increase in ⁸⁶Rb uptake but did not significantly alter Na⁺-K⁺-ATPase (ouabain-sensitive pNPPase) activity in crude membranes or cell sodium content. Taken together, the results point to a role for CCE and Akt phosphorylation, in response to low concentrations of ouabain, that increase the rate of cell proliferation without inhibiting Na⁺-K⁺-ATPase-mediated ion transport. Na⁺-K⁺-ATPase; opossum kidney cells     

Beta-subunit of cardiac Na+-K+-ATPase dictates the concentration of the functional enzyme in caveolae

Previous studies showed the presence of a significant fraction of Na⁺-K⁺-ATPase -subunits in cardiac myocyte caveolae, suggesting the caveolar interactions of Na⁺-K⁺-ATPase with its signaling partners. Because both - and -subunits are required for ATPase activity, to clarify the status of the pumping function of caveolar Na⁺-K⁺-ATPase, we have examined the relative distribution of two major subunit isoforms (₁ and ₁) in caveolar and noncaveolar membranes of adult rat cardiac myocytes. When cell lysates treated with high salt (Na₂CO₃ or KCl) concentrations were fractionated by a standard density gradient procedure, the resulting light caveolar membranes contained 30–40% of ₁-subunits and 80–90% of ₁-subunits. Use of Na₂CO₃ was shown to inactivate Na⁺-K⁺-ATPase; however, caveolar membranes obtained by the KCl procedure were not denatured and contained 75% of total myocyte Na⁺-K⁺-ATPase activity. Sealed isolated caveolae exhibited active Na⁺ transport. Confocal microscopy supported the presence of ,-subunits in caveolae, and immunoprecipitation showed the association of the subunits with caveolin oligomers. The findings indicate that cardiac caveolar inpocketings are the primary portals for active Na⁺-K⁺ fluxes, and the sites where the pumping and signaling functions of Na⁺-K⁺-ATPase are integrated. Preferential concentration of ₁-subunit in caveolae was cell specific; it was also noted in neonatal cardiac myocytes but not in fibroblasts and A7r5 cells. Uneven distributions of ₁ and ₁ in early and late endosomes of myocytes suggested different internalization routes of two subunits as a source of selective localization of active Na⁺-K⁺-ATPase in cardiac caveolae. cardiac myocyte; caveolin; oligomer; ouabain; sodium pump     

Alveolar epithelial fluid clearance persists in the presence of moderate left atrial hypertension in sheep

The effect of moderate left atrial(LA) hypertension on alveolar liquid clearance (ALC) wasinvestigated in anesthetized, ventilated sheep, surgically prepared tomeasure lung lymph flow as well as hemodynamics. To simulate alveolaredema, 3-4 ml/kg of isosmolar 5% albumin in Ringer lactate wereinstilled into each lower lobe, and ALC was measured. After 4 h of LAhypertension (24 cmH₂O), ALC wassimilar to that in control sheep (31 ± 3% with LA hypertension vs.34 ± 10% with normal LA pressure). Because plasma epinephrinelevels were moderately elevated in the presence of LA hypertension, ALCwas then studied in the presence of LA hypertension following bilateraladrenalectomy. Without endogenous release of epinephrine, ALC wassignificantly reduced compared with normal LA pressure (20 ± 7%compared with 34 ± 10%, P < 0.05). Thus endogenous catecholamines caused a submaximal stimulation of ALC in the presence of LA hypertension. Exogenous administration ofaerosolized ₂-agonist therapywith salmeterol increased ALC in the presence of normal LA pressure buthad no stimulatory effect in the presence of moderate LA hypertension.Therefore, we tested the hypothesis that endogenous release of atrialnatriuretic factor (ANF) may downregulate alveolar epithelialNa⁺ and fluid transport in thepresence of LA hypertension. There was a modest twofold increase inplasma ANF levels after LA hypertension. Additional in vitro studiesdemonstrated that, in the presence of₂-agonist stimulation, ANFdecreased Na⁺ pump activity(Na⁺-K⁺-ATPase)in isolated rat alveolar epithelial type II cells. ANF may downregulatevectorial Na⁺ and fluid transportstimulated by endogenous or exogenous -adrenergic agoniststimulation in the presence of LA hypertension. In summary, ALCcontinues even in the presence of moderate LA hypertension. Aerosolized₂-adrenergic agonist therapysignificantly increased ALC, but only when LA pressure was normal.

     

Effects of acute hyperoxic exposure on solute fluxes across the blood-gas barrier in rat lungs

Zheng, Lu P., Rui Sheng Du, and Barbara E. Goodman.Effects of acute hyperoxic exposure on solute fluxes across the blood-gas barrier in rat lungs. J. Appl.Physiol. 82(1): 240-247, 1997.We investigatedeffects of acute hyperoxia on solute transport from air space tovascular space in isolated rat lungs. Air spaces were filled withKrebs-Ringer bicarbonate solution containing fluoresceinisothiocyanate-labeled dextran (FD-20; mol wt 20,000) and either²²Na⁺and [¹⁴C]sucrose, orD-[¹⁴C]glucoseandL-[³H]glucose.Apparent permeability-surface area products for tracers over time (upto 120 min) were calculated for isolated perfused lungs from controlrats (room air) and rats exposed to >95%O₂ for 48 or 60 h immediatelypostexposure. After O₂ exposures,mean fluxes for[¹⁴C]sucrose and FD-20were significantly higher than in room-air control lungs. However,amiloride-sensitive Na⁺ and activeD-glucose fluxes were unchangedafter hyperoxic exposure. Therefore, it is unlikely that decreases innet solute transport in this lung-injury model contributed to pulmonaryedema resulting from O₂ toxicity.Increased net solute transport shown to help resolve pulmonary edemaafter acute hyperoxic exposure must therefore begin during the recoveryperiod. In summary, our data show increases in passive solute fluxesbut no changes in active solute fluxes immediately after acutehyperoxic lung injury.

     

Na+ influx and Na+-K+ pump activation during short-term exposure of cardiac myocytes to aldosterone

To examine the effect of aldosterone on sarcolemmalNa⁺ transport, we measuredouabain-sensitive electrogenicNa⁺-K⁺pump current(I_p) involtage-clamped ventricular myocytes and intracellularNa⁺ activity(aⁱ_Na) in right ventricularpapillary muscles. Aldosterone (10 nM) induced an increase in bothI_p and the rateof rise of aⁱ_Na duringNa⁺-K⁺pump blockade with the fast-acting cardiac steroid dihydroouabain. Thealdosterone-induced increase inI_p and rate ofrise of aⁱ_Na was eliminated bybumetanide, suggesting that aldosterone activates Na⁺ influx through theNa⁺-K⁺-2Clcotransporter. To obtain independent support for this, theNa⁺,K⁺, andCl concentrations in thesuperfusate and solution of pipettes used to voltage clamp myocyteswere set at levels designed to abolish the inward electrochemicaldriving force for theNa⁺-K⁺-2Clcotransporter. This eliminated the aldosterone-induced increase inI_p. We concludethat in vitro exposure of cardiac myocytes to aldosterone activates theNa⁺-K⁺-2Clcotransporter to enhance Na⁺influx and stimulate theNa⁺-K⁺pump.

     

Ammonium transport by the colonic H+-K+-ATPase expressed in Xenopus oocytes

Functional expression of the rat colonicH⁺-K⁺-ATPasewas obtained by coexpressing its catalytic -subunit and the₁-subunit of theNa⁺-K⁺-ATPasein Xenopus laevis oocytes. We observedthat, in oocytes expressing the rat colonicH⁺-K⁺-ATPasebut not in control oocytes (expressing₁ alone),NH₄Cl induced a decrease in⁸⁶Rb uptake and the initial rateof intracellular acidification induced by extracellularNH₄Cl was enhanced, consistentwith NH⁺₄ influx via the colonicH⁺-K⁺-ATPase.In the absence of extracellularK⁺, only oocytes expressing thecolonicH⁺-K⁺-ATPasewere able to acidify an extracellular medium supplemented withNH₄Cl. In the absence ofextracellular K⁺ and in thepresence of extracellular NH⁺₄, intracellular Na⁺ activity in oocytes expressingthe colonicH⁺-K⁺-ATPasewas lower than that in control oocytes. A kinetic analysis of⁸⁶Rb uptake suggests thatNH⁺₄ acts as a competitive inhibitor of thepump. Taken together, these results are consistent withNH⁺₄ competition forK⁺ on the external site of thecolonicH⁺-K⁺-ATPaseand with NH⁺₄ transport mediated by this pump.

     

九种常用杀虫剂对二化螟线粒体ATPase活力的抑制作用

研究了二化螟Chilo suppressalis线粒体Na⁺-K⁺-ATPase和Ca^2＋-Mg^2＋-ATPase的生物化学性质以及9种常用杀虫剂对这两种酶活性的影响。结果表明, 二化螟线粒体Na⁺-K⁺-ATPase和Ca^2＋-Mg^2＋-ATPase的最适反应条件为pH值7.4,温度37℃。 Na⁺-K⁺-ATPase的米氏常数（Km）为0.42 mmol/L,最大反应速度（Vmax）为302.47 nmol/(min·mg) 。Ca^2＋-Mg^2＋-ATPase的Km为0.40 mmol/L,Vmax为128.04 nmol/(min·mg)。药剂浓度为1×10^-4 mol/L时,5种菊酯类杀虫剂对离体ATPase活性抑制的顺序为：溴氰菊酯>联苯菊酯>百树菊酯>三氟氯氰菊酯和氟硅菊酯;对二化螟Na⁺-K⁺-ATPase的抑制率分别为40.12％、39.69％、27.27％、19.49％和18.71％;对Ca^2＋-Mg^2＋-ATPase的抑制率分别为29.27％、23.78％、19.88％、11.64%和14.34％。硫丹对二化螟Na⁺-K⁺-ATPase和Ca^2＋-Mg^2＋-ATPase的抑制率均为17.46％。甲胺磷和呋喃丹对Ca^2＋-Mg^2＋-ATPase的抑制率分别为27.16％和17.42％,对Na⁺-K⁺-ATPase则几乎没有抑制作用。实验结果还表明, 在1.6×10^-7～1×10^-4 mol/L的浓度范围内,上述9种杀虫剂对二化螟ATPase活性的抑制率存在明显的剂量-效应关系。     

Distribution of sodium transporters and aquaporin-1 in the human choroid plexus

The choroid plexus epithelium secretes electrolytes and fluid in the brain ventricular lumen at high rates. Several channels and ion carriers have been identified as likely mediators of this transport in rodent choroid plexus. This study aimed to map several of these proteins to the human choroid plexus. Immunoperoxidase-histochemistry was employed to determine the cellular and subcellular localization of the proteins. The water channel, aquaporin (AQP) 1, was predominantly situated in the apical plasma membrane domain, although distinct basolateral and endothelial immunoreactivity was also observed. The Na⁺-K⁺-ATPase ₁-subunit was exclusively localized apically in the human choroid plexus epithelial cells. Immunoreactivity for the Na⁺-K⁺-2Cl^– cotransporter, NKCC1, was likewise confined to the apical plasma membrane domain of the epithelium. The Cl^–/HCO₃^– exchanger, AE2, was localized basolaterally, as was the Na⁺-dependent Cl^–/HCO₃^– exchanger, NCBE, and the electroneutral Na⁺-HCO₃^– cotransporter, NBCn1. No immunoreactivity was found toward the Na⁺-dependent acid/base transporters NHE1 or NBCe2. Hence, the human choroid plexus epithelium displays an almost identical distribution pattern of water channels and Na⁺ transporters as the rat and mouse choroid plexus. This general cross species pattern suggests central roles for these transporters in choroid plexus functions such as cerebrospinal fluid production. immunohistochemistry; metabolism; cerebrospinal fluid secretion     

Heterologous expression of Na(+)-K(+)-ATPase in insect cells: intracellular distribution of pump subunits

TheNa⁺-K⁺-ATPase is a heterodimeric plasmamembrane protein responsible for cellular ionic homeostasis in nearlyall animal cells. It has been shown that some insect cells (e.g., HighFive cells) have no (or extremely low)Na⁺-K⁺-ATPase activity. We expressed sheepkidney Na⁺-K⁺-ATPase - and -subunitsindividually and together in High Five cells via the baculovirusexpression system. We used quantitative slot-blot analyses to determinethat the expressed Na⁺-K⁺-ATPase comprisesbetween 0.5% and 2% of the total membrane protein in these cells.Using a five-step sucrose gradient (0.8-2.0 M) to separate theendoplasmic reticulum, Golgi apparatus, and plasma membrane fractions,we observed functional Na⁺ pump molecules in each membranepool and characterized their properties. Nearly all of the expressedprotein functions normally, similar to that found in purified dogkidney enzyme preparations. Consequently, the measurements describedhere were not complicated by an abundance of nonfunctionalheterologously expressed enzyme. Specifically, ouabain-sensitive ATPaseactivity, [³H]ouabain binding, and cation dependencieswere measured for each fraction. The functional properties of theNa⁺-K⁺-ATPase were essentially unaltered afterassembly in the endoplasmic reticulum. In addition, we measuredouabain-sensitive ⁸⁶Rb⁺ uptake in whole cellsas a means to specifically evaluateNa⁺-K⁺-ATPase molecules that were properlyfolded and delivered to the plasma membrane. We could not measure anyouabain-sensitive activities when either the -subunit or -subunitwere expressed individually. Immunostaining of the separate membranefractions indicates that the -subunit, when expressed alone, isdegraded early in the protein maturation pathway (i.e., the endoplasmicreticulum) but that the -subunit is processed normally and deliveredto the plasma membrane. Thus it appears that only the -subunit hasan oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the - heterodimer within theendoplasmic reticulum apparently does not require a Na⁺pump-specific chaperone.