Epidermal growth factor increases lung liquid clearance in rat lungs

Epidermal growthfactor (EGF) has been reported to stimulate the proliferation ofepithelial cells and increase Na⁺flux andNa⁺-K⁺-ATPasefunction in alveolar epithelial cell monolayers. Increases inNa⁺-K⁺-ATPasein alveolar type II cells (AT2) have been associated with increasedactive Na⁺ transport and lungedema clearance across the rat alveolar epithelium in a model ofproliferative lung injury. Thus we tested whether administration ofaerosolized EGF to rat lungs would increase activeNa⁺ transport and lung liquidclearance. Sixteen adult Sprague-Dawley male rats were randomized tothree groups. To a group of six rats, an aerosol generated from 20 µgof EGF in saline was delivered to the lungs, to a second group of fiverats only aerosolized saline was delivered, and a third group of fiverats without treatment served as the control. Forty-eight hourspostaerosolization of rat lungs with EGF there was an ~40% increasein active Na⁺ transport and lungliquid clearance compared with control rats, in the absence of changesin²²Na⁺,[³H]mannitol, andalbumin permeabilities. TheNa⁺-K⁺-ATPaseactivity in AT2 cells harvested from these lungs was increased in ratsthat received aerosolized EGF compared with AT2 cells from both controlrats and rats receiving aerosolized saline. These results support thehypothesis that in vivo delivery of EGF aerosols upregulates alveolarepithelialNa⁺-K⁺-ATPaseand increases lung liquid clearance in rats.

     

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.

     

Functional demonstration of Na+-K+-2Cl- cotransporter activity in isolated, polarized choroid plexus cells

The function of the apicalNa⁺-K⁺-2Clcotransporter in mammalian choroid plexus (CP) is uncertain andcontroversial. To investigate cotransporter function, we developed anovel dissociated rat CP cell preparation in which single, isolatedcells maintain normal polarized morphology. Immunofluorescencedemonstrated that in isolated cells theNa⁺-K⁺-ATPase,Na⁺-K⁺-2Clcotransporter, and aquaporin 1 water channel remained localized to thebrush border, whereas theCl/HCO₃(anion) exchanger type 2 was confined to the basolateral membrane. Weutilized video-enhanced microscopy and cell volume measurementtechniques to investigate cotransporter function. Application of 100 µM bumetanide caused CP cells to shrink rapidly. Elevation ofextracellular K⁺ from 3 to 6 or 25 mM caused CP cells to swell 18 and 33%, respectively. Swelling wasblocked completely by Na⁺ removalor by addition of 100 µM bumetanide. Exposure of CP cells to 5 mMBaCl₂ induced rapid swelling thatwas inhibited by 100 µM bumetanide. We conclude that the CPcotransporter is constitutively active and propose that it functions inseries with Ba²⁺-sensitiveK⁺ channels to reabsorbK⁺ from cerebrospinal fluid to blood.

     

Sch-28080 depletes intracellular ATP selectively in mIMCD-3 cells

Two H⁺-K⁺-ATPase isoforms are presentin kidney: the gastric, highly sensitive to Sch-28080, and the colonic,partially sensitive to ouabain. Upregulation of Sch-28080-sensitiveH⁺-K⁺-ATPase, or "gastric"H⁺-K⁺-ATPase, has been demonstrated inhypokalemic rat inner medullary collecting duct cells (IMCDs).Nevertheless, only colonic H⁺-K⁺-ATPase mRNAand protein abundance increase in this condition. This study wasdesigned to determine whether Sch-28080 inhibits transporters otherthan the gastric H⁺-K⁺-ATPase. In the presenceof bumetanide, Sch-28080 (200 µM) and ouabain (2 mM) inhibited⁸⁶Rb⁺ uptake (>90%). That⁸⁶Rb⁺ uptake was almost completely abolished bySch-28080 indicates an effect of this agent on theNa⁺-K⁺-ATPase. ATPase assays in membranes, orlysed cells, demonstrated sensitivity to ouabain but not Sch-28080.Thus the inhibitory effect of Sch-28080 was dependent on cellintegrity. ⁸⁶Rb⁺-uptake studies withoutbumetanide demonstrated that ouabain inhibited activity by only50%. Addition of Sch-28080 (200 µM) blocked all residualactivity. Intracellular ATP declined after Sch-28080 (200 µM) butrecovered after removal of this agent. In conclusion, highconcentrations of Sch-28080 inhibit K⁺-ATPase activity inmouse IMCD-3 (mIMCD-3) cells as a result of ATP depletion.

     

Na+-K+-2Cl- cotransport in Ehrlich cells: regulation by protein phosphatases and kinases

To identify protein kinases (PK) and phosphatases (PP) involvedin regulation of theNa⁺-K⁺-2Clcotransporter in Ehrlich cells, the effect of various PK and PPinhibitors was examined. The PP-1, PP-2A, and PP-3 inhibitor calyculinA (Cal-A) was a potent activator ofNa⁺-K⁺-2Clcotransport (EC₅₀ = 35 nM).Activation by Cal-A was rapid (<1 min) but transient. Inactivation isprobably due to a 10% cell swelling and/or the concurrentincrease in intracellularCl concentration. Cellshrinkage also activates theNa⁺-K⁺-2Clcotransport system. Combining cell shrinkage with Cal-A treatment prolonged the cotransport activation compared with stimulation withCal-A alone, suggesting PK stimulation by cell shrinkage. Shrinkage-induced cotransport activation was pH andCa²⁺/calmodulin dependent.Inhibition of myosin light chain kinase by ML-7 and ML-9 or of PKA byH-89 and KT-5720 inhibited cotransport activity induced by Cal-A and bycell shrinkage, with IC₅₀ values similar to reported inhibition constants of the respective kinases invitro. Cell shrinkage increased the ML-7-sensitive cotransport activity, whereas the H-89-sensitive activity was unchanged, suggesting that myosin light chain kinase is a modulator of theNa⁺-K⁺-2Clcotransport activity during regulatory volume increase.

     

Contractile regulation of the Na+-K+-2Cl{-} cotransporter in vascular smooth muscle

Vasoconstrictors activate theNa⁺-K⁺-2Cl cotransporter NKCC1 inrat aortic smooth muscle, but the mechanism is unknown. Efflux of⁸⁶Rb⁺ from rat aorta in response tophenylephrine (PE) was measured in the absence and presence ofbumetanide, a specific inhibitor of NKCC1. Removal of extracellularCa²⁺ completely abolished the activation of NKCC1 by PE.This was not due to inhibition of Ca²⁺-dependentK⁺ channels since blocking these channels withBa²⁺ in Ca²⁺-replete solution did not preventactivation of NKCC1 by PE. Stimulation of NKCC1 by PE was inhibited70% by 75 µM ML-9, 97% by 2 µM wortmannin, and 70% by 2 mM2,3-butanedione monoxime, each of which inhibited isometric forcegeneration in aortic rings. Bumetanide-insensitive Rb⁺efflux, an indication of Ca²⁺-dependent K⁺channel activity, was reduced by ML-9 but not by the other inhibitors. Stretching of aortic rings on tubing to increase lumen diameter to120% of normal almost completely blocked the stimulation of NKCC1 byPE without inhibiting the stimulation by hypertonic shrinkage. Weconclude that activation of theNa⁺-K⁺-2Cl cotransporter by PE isthe direct result of smooth muscle contraction throughCa²⁺-dependent activation of myosin light chain kinase.This indicates that theNa⁺-K⁺-2Cl cotransporter isregulated by the contractile state of vascular smooth muscle.

     

Vasoconstrictors and nitrovasodilators reciprocally regulate the Na+-K+-2Cl- cotransporter in rat aorta

Little is knownabout the function and regulation of theNa⁺-K⁺-2Clcotransporter NKCC1 in vascular smooth muscle. Theactivity of NKCC1 was measured as the bumetanide-sensitive efflux of⁸⁶Rb⁺from intact smooth muscle of the rat aorta. Hypertonic shrinkage (440 mosmol/kgH₂O) rapidlydoubled cotransporter activity, consistent with its volume-regulatoryfunction. NKCC1 was also acutely activated by the vasoconstrictors ANGII (52%), phenylephrine (50%), endothelin (53%), and 30 mM KCl(54%). Both nitric oxide and nitroprusside inhibited basal NKCC1activity (39 and 34%, respectively), and nitroprussidecompletely reversed the stimulation by phenylephrine. Thephosphorylation of NKCC1 was increased by hypertonic shrinkage, phenylephrine, and KCl and was reduced by nitroprusside. The inhibition of NKCC1 significantly reduced the contraction of rat aorta induced byphenylephrine (63% at 10 nM, 26% at 30 nM) but not by KCl. Weconclude that theNa⁺-K⁺-2Clcotransporter in vascular smooth muscle is reciprocally regulated byvasoconstrictors and nitrovasodilators and contributes to smooth musclecontraction, indicating that alterations in NKCC1 could influencevascular smooth muscle tone in vivo.

     

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⁺].

     

Carbachol inhibits Na(+)-K(+)-ATPase activity in choroid plexus via stimulation of the NO/cGMP pathway

Secretion of cerebrospinal fluid by the choroid plexus canbe inhibited by its cholinergic innervation. We demonstrated that carbachol inhibits the Na⁺-K⁺-ATPase in bovinechoroid tissue slices and investigated the mechanism. Many of theactions of cholinergic agents are mediated by nitric oxide (NO), whichplays important roles in fluid homeostasis. The inhibition ofNa⁺-K⁺-ATPase was blocked by the NO synthaseinhibitor [N-nitro-L-argininemethyl ester] and was quantitatively mimicked by the NO agonistssodium nitroprusside (SNP) and diethylenetriamine NO. Inhibition by SNPcorrelated with an increase in tissue cGMP and was abolished by1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase. Inhibition was mimicked bythe protein kinase G activator 8-bromo-cGMP and by okadaic acid, aninhibitor of protein phosphatases 1 and 2A. cGMP-dependent proteinkinase inhibitors Rp-8-pCPT-cGMP (0.5-5 µM) and KT-5823 (2.0 µM) did not block the effects of SNP, but higher concentrations ofthe more selective inhibitor (Rp-8-pCPT-cGMP) had a pharmacological inhibitory effect on Na⁺-K⁺-ATPase. The datasuggest that cholinergic regulation of theNa⁺-K⁺-ATPase is mediated by NO and involvesactivation of guanylate cyclase and elevation of cGMP.

     

Ni2+ transport by the human Na+/Ca2+ exchanger expressed in Sf 9cells

The mechanism ofNi²⁺ block of theNa⁺/Ca²⁺exchanger was examined in Sf 9 cells expressing the human heartNa⁺/Ca²⁺exchanger (NCX1-NACA1). As predicted from the reported actions ofNi²⁺, its application reducedextracellular Na⁺-dependentchanges in intracellular Ca²⁺concentration (measured by fluo 3 fluorescence changes). However, contrary to expectation, the reduced fluorescence was accompanied bymeasured⁶³Ni²⁺entry. The⁶³Ni²⁺entry was observed in Sf 9 cells expressing theNa⁺/Ca²⁺exchanger but not in control cells. The established sequential transport mechanism of theNa⁺/Ca²⁺exchanger could be compatible with these results if one of the two iontranslocation steps is blocked byNi²⁺ and the other permitsNi²⁺ translocation. We concludethat, because Ni²⁺ entry wasinhibited by extracellular Ca²⁺and enhanced by extracellular Na⁺,the Ca²⁺ translocation step movedNi²⁺, whereas theNa⁺ translocation step wasinhibited by Ni²⁺. A model ispresented to discuss these findings.     

Na+-K+-ATPase in frog esophagus mucociliary cell membranes: inhibition by protein kinase C activation

We examined protein kinase C (PKC)-dependentregulation ofNa⁺-K⁺-ATPasein frog mucociliary cells. Activation of PKC by12-O-tetradecanoylphorbol-13-acetate (TPA) or 1,2-dioctanoyl-sn-glycerol(diC8) either in intact cells or isolated membranes resulted in aspecific inhibition ofNa⁺-K⁺-ATPaseactivity by ~25-45%. The inhibitory effects in membranes exhibited time dependence and dose dependence [half-maximalinhibition concentration (IC₅₀) = 0.5 ± 0.1 nM and 2.4 ± 0.2 µM, respectively, for TPA anddiC8] and were not influenced byCa²⁺. Analysis of the ouabaininhibition pattern revealed the presence of twoNa⁺-K⁺-ATPaseisoforms with IC₅₀ values forcardiac glycoside of 2.6 ± 0.8 nM and 409 ± 65 nM,respectively. Most importantly, the isoform possessing a higheraffinity for ouabain was almost completely inhibited by TPA, whereasits counterpart was hardly sensitive to the PKC activator. The resultssuggest that, in frog mucociliary cells, PKC regulatesNa⁺-K⁺-ATPaseand that this action is related to the specificNa⁺-K⁺-ATPaseisoform.

     

Regional distribution of the Na+ and K+ currents around the crystalline lens of rabbit

Early studies described asymmetricalelectrical properties across the ocular lens in theanterior-to-posterior direction. More recent results obtained with avibrating probe indicated that currents around the lens surface are notuniform by showing an outwardly directed K⁺ efflux at thelens equator and Na⁺ influx at the poles. The latterstudies have been used to support theoretical models for fluidrecirculation within the avascular lens. However, the existence of anonuniform current distribution in the lens epithelium from theanterior pole to the equator has never been confirmed. The present workdeveloped a modified short-circuiting technique to examine the netflows of Na⁺ and K⁺ across arbitrarily definedlens surface regions. Results indicate that passive inflows ofNa⁺ occur at both the anterior polar region and posteriorlens surface, consistent with suggestions derived from the vibratingprobe data, whereas K⁺ efflux plus theNa⁺-K⁺ pump-generated current comprise thecurrents at the equatorial surface and an area anterior to it.Furthermore, Na⁺-K⁺ pump activity was absent atthe posterior surface and its polar region in all lenses examined, aswell as from the anterior polar region in most lenses. The latterunexpected observation suggests that the monolayered epithelium, whichis confined to the anterior surface of the lens, does not express anactive Na⁺-K⁺ pump at its anterior-most aspect.Nevertheless, this report represents the first independent confirmationthat positive currents leave the lens around the equator and reenteracross the polar and posterior surfaces.

     

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     

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.

     

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.

     

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.

     

Regulation of Na(+)-K(+)-Cl(-) cotransporter in primary astrocytes by dibutyryl cAMP and high [K(+)](o)

In this study, we examined theNa⁺-K⁺-Cl cotransporter activityand expression in rat cortical astrocyte differentiation. Astrocyte differentiation was induced by dibutyryl cAMP (DBcAMP, 0.25 mM) for7 days, and cells changed from a polygonal to process-bearing morphology. Basal activity of the cotransporter was significantly increased in DBcAMP-treated astrocytes (P < 0.05).Expression of an ~161-kDa cotransporter protein was increased by 91%in the DBcAMP-treated astrocytes. Moreover, the specific[³H]bumetanide binding was increased by 67% in theDBcAMP-treated astrocytes. Inhibition of protein synthesis bycyclohexamide (2-3 µg/ml) significantly attenuated theDBcAMP-mediated upregulation of the cotransporter activity andexpression. The Na⁺-K⁺-Clcotransporter in astrocytes has been suggested to play a role inK⁺ uptake. In 75 mM extracellular K⁺concentration, the cotransporter-mediated K⁺ influx wasstimulated by 147% in nontreated cells and 79% in DBcAMP-treatedcells (P < 0.05). To study whether this highK⁺-induced stimulation of the cotransporter is attributedto membrane depolarization and Ca²⁺ influx, the role of theL-type voltage-dependent Ca²⁺ channel was investigated. Thehigh-K⁺-mediated stimulation of the cotransporter activitywas abolished in the presence of either 0.5 or 1.0 µM of the L-typechannel blocker nifedipine or Ca²⁺-free HEPES buffer. Arise in intracellular free Ca²⁺ in astrocytes was observedin high K⁺. These results provide the first evidence thatthe Na⁺-K⁺-Cl cotransporterprotein expression can be regulated selectively when intracellular cAMPis elevated. The study also demonstrates that the cotransporter inastrocytes is stimulated by high K⁺ in aCa²⁺-dependent manner.

     

Stimulation of Na+-K+-2Cl- cotransporter in neuronal cells by excitatory neurotransmitter glutamate

Na⁺-K⁺-2Clcotransporters are important in renal salt reabsorption and in saltsecretion by epithelia. They are also essential in maintenance andregulation of ion gradients and cell volume in both epithelial andnonepithelial cells. Expression ofNa⁺-K⁺-2Clcotransporters in brain tissues is high; however, little is known abouttheir function and regulation in neurons. In this study, we examinedregulation of theNa⁺-K⁺-2Clcotransporter by the excitatory neurotransmitter glutamate. The cotransporter activity in human neuroblastoma SH-SY5Y cells was assessed by bumetanide-sensitiveK⁺ influx, and protein expressionwas evaluated by Western blot analysis. Glutamate was found to induce adose- and time-dependent stimulation ofNa⁺-K⁺-2Clcotransporter activity in SH-SY5Y cells. Moreover, both the glutamate ionotropic receptor agonistN-methyl-D-asparticacid (NMDA) and the metabotropic receptor agonist(±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) significantlystimulated the cotransport activity in these cells.NMDA-mediated stimulation of theNa⁺-K⁺-2Clcotransporter was abolished by the selective NMDA-receptor antagonist (+)-MK-801 hydrogen maleate.trans-ACPD-mediated effect on the cotransporter was blocked by the metabotropic receptor antagonist (+)--methyl-(4-carboxyphenyl)glycine. The results demonstrate thatNa⁺-K⁺-2Clcotransporters in neurons are regulated by activation of both ionotropic and metabotropic glutamate receptors.

     

Regulation of Na(+)-K(+)-ATPase by cAMP-dependent protein kinase anchored on membrane via its anchoring protein

Na⁺-K⁺- ATPase -subunitsin basolateral membrane vesicles (BLMVs) purified from rat parotidglands were ³²P-labeled within 5 s by incubation with[-³²P]ATP at 37°C in the presence of cAMP, but nolabeling occurred without cAMP. Phosphorylation ofNa⁺-K⁺-ATPase was associated with a decrease inits activity. This -subunit phosphorylation disappeared when BLMVswere briefly incubated with cAMP and subsequent washing before theincubation with [-³²P]ATP, indicating that catalyticsubunit of protein kinase A (PKA) associated to BLMVs via binding withits RII regulatory subunit anchored on the membrane. In theabsence of cAMP, a PKA catalytic subunit readily reassociated with themembrane-bound RII subunit. HT-31 peptide inhibited theNa⁺-K⁺-ATPase phosphorylation by membrane-boundendogenous PKA, indicating an involvement of A-kinase anchoring protein(AKAP). AKAP-150 protein in BLMVs was shown by immunoblotting and anRII overlay assay and was coimmunoprecipitated by anti-RII antibody.These results show that Na⁺-K⁺-ATPase of ratparotid gland acinar cells is regulated in vivo by membrane-anchoredPKA via AKAP rather than by free cytosolic PKA.

     

Incubation of OKP cells in low-K+ media increases NHE3 activity after early decrease in intracellular pH

Chronichypokalemia increases the activity of proximal tubule apical membraneNa⁺/H⁺antiporter NHE3. The present study examined the effect ofthe incubation of OKP cells (an opossum kidney, clone P cell line) incontrol medium {K⁺ concn([K⁺]) = 5.4 mM} or low-K⁺ medium([K⁺] = 2.7 mM) onNHE3. The activity of an ethylisopropyl amiloride-resistant Na⁺/H⁺antiporter, whose characteristics were consistent with those ofNHE3, was increased inlow-K⁺ cells beginning at 8 h.NHE3 mRNA and NHE3 protein abundance were increased 2.2-fold and 62%,respectively, at 24 h but not at 8 h. After incubation inlow-K⁺ medium, intracellular pH(pH_i) decreased by 0.27 pH units(maximum at 27 min) and then recovered to the control level.Intracellular acidosis induced by 5 mM sodium propionate increasedNa⁺/H⁺antiporter activity at 8 and 24 h. Herbimycin A, a tyrosine kinase inhibitor, blocked low-K⁺- andsodium propionate-induced activation of theNa⁺/H⁺antiporter at 8 and 24 h. Our results demonstrate thatlow-K⁺ medium causes an earlydecrease in pH_i, which leads to anincrease in NHE3 activity via a tyrosine kinase pathway.