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1.
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.

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2.
We examined the effects of human cytomegalovirus (HCMV)infection on theNa+-K+-Clcotransporter (NKCC) in a human fibroblast cell line. Using the Cl-sensitive dye MQAE, weshowed that the mock-infected MRC-5 cells express a functional NKCC.1) IntracellularCl concentration([Cl]i)was significantly reduced from 53.4 ± 3.4 mM to 35.1 ± 3.6 mMfollowing bumetanide treatment. 2)Net Cl efflux caused byreplacement of external Clwith gluconate was bumetanide sensitive.3) InCl-depleted mock-infectedcells, the Cl reuptake rate(in HCO3-free media) was reduced inthe absence of external Na+ and bytreatment with bumetanide. After HCMV infection, we found that although[Cl]iincreased progressively [24 h postexposure (PE), 65.2 ± 4.5 mM; 72 h PE, 80.4 ± 5.0 mM], the bumetanide andNa+ sensitivities of[Cl]iand net Cl uptake and losswere reduced by 24 h PE and abolished by 72 h PE. Western blots usingthe NKCC-specific monoclonal antibody T4 showed an approximatelyninefold decrease in the amount of NKCC protein after 72 h ofinfection. Thus HCMV infection resulted in the abolition of NKCCfunction coincident with the severe reduction in the amount of NKCCprotein expressed.

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3.
Mercury alters thefunction of proteins by reacting with cysteinyl sulfhydryl(SH) groups. Theinorganic form (Hg2+) is toxicto epithelial tissues and interacts with various transport proteinsincluding the Na+ pump andCl channels. In this study,we determined whether theNa+-K+-Clcotransporter type 1 (NKCC1), a major ion pathway in secretory tissues,is also affected by mercurial substrates. To characterize theinteraction, we measured the effect ofHg2+ on ion transport by thesecretory shark and human cotransporters expressed in HEK-293 cells.Our studies show that Hg2+inhibitsNa+-K+-Clcotransport, with inhibitor constant(Ki) values of25 µM for the shark carrier (sNKCC1) and 43 µM for thehuman carrier. In further studies, we took advantage of speciesdifferences in Hg2+ affinity toidentify residues involved in the interaction. An analysis ofhuman-shark chimeras and of an sNKCC1 mutant(Cys-697Leu) reveals that transmembrane domain 11 plays an essential role in Hg2+binding. We also show that modification of additionalSH groups by thiol-reactingcompounds brings about inhibition and that the binding sites are notexposed on the extracellular face of the membrane.

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4.
The purpose ofthe current experiments was 1) toassess basolateralNa+-K+-2Clcotransporter (NKCC1) expression and2) to ascertain the role of cysticfibrosis transmembrane conductance regulator (CFTR) in the regulationof this transporter in a prototypical pancreatic duct epithelial cellline. Previously validated human pancreatic duct celllines (CFPAC-1), which exhibit physiological features prototypical ofcystic fibrosis, and normal pancreatic duct epithelia (stablerecombinant CFTR-bearing CFPAC-1 cells, termed CFPAC-WT) were grown toconfluence before molecular and functional studies. High-stringencyNorthern blot hybridization, utilizing specific cDNA probes, confirmedthat NKCC1 was expressed in both cell lines and its mRNA levels weretwofold higher in CFPAC-WT cells than in CFPAC-1 cells(P < 0.01, n = 3).Na+-K+-2Clcotransporter activity, assayed as the bumetanide-sensitive, Na+- andCl-dependentNH+4 entry into the cell (withNH+4 acting as a substitute forK+), increased by ~115% inCFPAC-WT cells compared with CFPAC-1 cells(P < 0.01, n = 6). Reducing the intracellularCl by incubating the cellsin a Cl-free mediumincreasedNa+-K+-2Clcotransporter activity by twofold (P < 0.01, n = 4) only in CFPAC-WT cells. We concluded that NKCC1 is expressed in pancreatic duct cellsand mediates the entry ofCl. NKCC1 activity isenhanced in the presence of an inwardCl gradient. The resultsfurther indicate that the presence of functional CFTR enhances theexpression of NKCC1. We speculate that CFTR regulates this process in aCl-dependent manner.

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5.
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.

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6.
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.

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7.
A reduction in angiotensinII (ANG II) in vivo by treatment of rabbits with theangiotensin-converting enzyme inhibitor, captopril, increasesNa+-K+ pump current (Ip)of cardiac myocytes. This increase is abolished by exposure of myocytesto ANG II in vitro. Because ANG II induces translocation of the-isoform of protein kinase C (PKC), we examined whether thisisozyme regulates the pump. We treated rabbits with captopril, isolatedmyocytes, and measured Ip of myocytes voltageclamped with wide-tipped patch pipettes. Ip ofmyocytes from captopril-treated rabbits was larger thanIp of myocytes from controls. ANG II superfusionof myocytes from captopril-treated rabbits decreasedIp to levels similar to controls. Inclusion ofPKC-specific blocking peptide in pipette solutions used to perfusethe intracellular compartment abolished the effect of ANG II. Inclusionof RACK, a PKC-specific activating peptide, in pipettesolutions had an effect on Ip that was similarto that of ANG II. There was no additive effect of ANG II andRACK. We conclude that PKC regulates the sarcolemmalNa+-K+ pump.

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8.
Previous data indicate that adenosine 3',5'-cyclicmonophosphate activates the epithelial basolateralNa+-K+-Clcotransporter in microfilament-dependent fashion in part by direct action but also in response to apicalCl loss (due to cellshrinkage or decreased intracellularCl). To further addressthe actin dependence ofNa+-K+-Clcotransport, human epithelial T84 monolayers were exposed to anisotonicity, and isotopic flux analysis was performed.Na+-K+-Clcotransport was activated by hypertonicity induced by added mannitol but not added NaCl. Cotransport was also markedly activated by hypotonic stress, a response that appeared to be due in part to reduction of extracellularCl concentration and alsoto activation of K+ andCl efflux pathways.Stabilization of actin with phalloidin blunted cotransporter activationby hypotonicity and abolished hypotonic activation ofK+ andCl efflux. However,phalloidin did not prevent activation of cotransport by hypertonicityor isosmotic reduction of extracellularCl. Conversely, hypertonicbut not hypotonic activation was attenuated by the microfilamentdisassembler cytochalasin D. The results emphasize the complexinterrelationship among intracellularCl activity, cell volume,and the actin cytoskeleton in the regulation of epithelialCl transport.

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9.
Data obtainedduring the last two decades show that spontaneously hypertensive rats,an acceptable experimental model of primary human hypertension, possessincreased activity of both ubiquitous and renal cell-specific isoformsof theNa+/H+exchanger (NHE) andNa+-K+-2Clcotransporter. Abnormalities of these ion transporters have been foundin patients suffering from essential hypertension. Recent geneticstudies demonstrate that genes encoding the - and -subunits ofENaC, a renal cell-specific isoform of theNa+-K+-2Clcotransporter, and 3-, 1-, and 2-subunits of theNa+-K+pump are localized within quantitative trait loci (QTL) for elevated blood pressure as well as for enhanced heart-to-body weight ratio, proteinuria, phosphate excretion, and stroke latency. On the basis ofthe homology of genome maps, several other genes encoding these transporters, as well as theNa+/H+exchanger andNa+-K+-2Clcotransporter, can be predicted in QTL related to the pathogenesis ofhypertension. However, despite their location within QTL, analysis ofcDNA structure did not reveal any mutation in the coding region of theabove-listed transporters in primary hypertension, with the exceptionof G276L substitution in the1-Na+-K+pump from Dahl salt-sensitive rats and a higher occurrence of T594Mmutation of -ENaC in the black population with essential hypertension. These results suggest that, in contrast to Mendelian forms of hypertension, the altered activity of monovalent ion transporters in primary hypertension is caused by abnormalities ofsystems involved in the regulation of their expression and/or function.Further analysis of QTL in F2hybrids of normotensive and hypertensive rats and in affected siblingpairs will allow mapping of genes causing abnormalities ofthese regulatory pathways.  相似文献   

10.
We reported previously that inhibition ofNa+-K+-Cl cotransporter isoform 1 (NKCC1) by bumetanide abolishes high extracellular K+concentration ([K+]o)-induced swelling andintracellular Cl accumulation in rat cortical astrocytes.In this report, we extended our study by using cortical astrocytes fromNKCC1-deficient (NKCC1/) mice. NKCC1 protein andactivity were absent in NKCC1/ astrocytes.[K+]o of 75 mM increased NKCC1 activityapproximately fourfold in NKCC1+/+ cells (P < 0.05) but had no effect in NKCC1/ astrocytes.Intracellular Cl was increased by 70% inNKCC1+/+ astrocytes under 75 mM[K+]o (P < 0.05) butremained unchanged in NKCC1/ astrocytes. Baselineintracellular Na+ concentration([Na+]i) in NKCC1+/+ astrocyteswas 19.0 ± 0.5 mM, compared with 16.9 ± 0.3 mM[Na+]i in NKCC1/ astrocytes(P < 0.05). Relative cell volume ofNKCC1+/+ astrocytes increased by 13 ± 2% in 75 mM[K+]o, compared with a value of 1.0 ± 0.5% in NKCC1/ astrocytes (P < 0.05).Regulatory volume increase after hypertonic shrinkage was completelyimpaired in NKCC1/ astrocytes.High-[K+]o-induced 14C-labeledD-aspartate release was reduced by ~30% inNKCC1/ astrocytes. Our study suggests that stimulationof NKCC1 is required for high-[K+]o-inducedswelling, which contributes to glutamate release from astrocytes underhigh [K+]o.

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11.
Cell pH was monitored in medullary thick ascending limbs todetermine effects of ANG II onNa+-K+(NH+4)-2Clcotransport. ANG II at 1016to 1012 M inhibited30-50% (P < 0.005),but higher ANG II concentrations were stimulatory compared with the1012 M ANG II levelcotransport activity; eventually,106 M ANG II stimulated34% cotransport activity (P < 0.003). Inhibition by 1012M ANG II was abolished by phospholipase C (PLC), diacylglycerol lipase,or cytochrome P-450-dependentmonooxygenase blockade; 1012 M ANG II had no effectadditive to inhibition by 20-hydroxyeicosatetranoic acid (20-HETE).Stimulation by 106 M ANG IIwas abolished by PLC and protein kinase C (PKC) blockade and waspartially suppressed when the rise in cytosolicCa2+ was prevented. All ANG IIeffects were abolished by DUP-753 (losartan) but not by PD-123319. Thus1012 M ANG II inhibitsvia 20-HETE, whereas 5 × 1011 M ANG II stimulatesvia PKCNa+-K+(NH+4)-2Clcotransport; all ANG II effects involveAT1 receptors and PLC activation.

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12.
The role of the Na+ pump2-subunit in Ca2+ signaling was examined inprimary cultured astrocytes from wild-type(2+/+ = WT) mouse fetuses and thosewith a null mutation in one [2+/ = heterozygote (Het)] or both [2/ = knockout (KO)] 2 genes. Na+ pump catalytic() subunit expression was measured by immunoblot; cytosol[Na+] ([Na+]cyt) and[Ca2+] ([Ca2+]cyt) weremeasured with sodium-binding benzofuran isophthalate and fura 2 byusing digital imaging. Astrocytes express Na+ pumpswith both 1- (80% of total ) and2- (20% of total ) subunits. Het astrocytesexpress 50% of normal 2; those from KO express none.Expression of 1 is normal in both Het and KO cells.Resting [Na+]cyt = 6.5 mM in WT, 6.8 mMin Het (P > 0.05 vs. WT), and 8.0 mM in KO cells(P < 0.001); 500 nM ouabain (inhibits only2) equalized [Na+]cyt at 8 mMin all three cell types. Resting[Ca2+]cyt = 132 nM in WT, 162 nM in Het,and 196 nM in KO cells (both P < 0.001 vs. WT).Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER)Ca2+ pumps and unloads the ER, induces transient (inCa2+-free media) or sustained (in Ca2+-repletemedia) elevation of [Ca2+]cyt. TheseCa2+ responses to 10 µM CPA were augmented in Het as wellas KO cells. When CPA was applied in Ca2+-free media, thereintroduction of Ca2+ induced significantly largertransient rises in [Ca2+]cyt (due toCa2+ entry through store-operated channels) in Het and KOcells than in WT cells. These results correlate with published evidencethat 2 Na+ pumps andNa+/Ca2+ exchangers are confined to plasmamembrane microdomains that overlie the ER. The data suggest thatselective reduction of 2 Na+ pump activitycan elevate local [Na+] and, viaNa+/Ca2+ exchange, [Ca2+] in thetiny volume of cytosol between the plasma membrane and ER. This, inturn, augments adjacent ER Ca2+ stores and therebyamplifies Ca2+ signaling without elevating bulk[Na+]cyt.

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13.
Alveolar epithelial cells were isolated from adultSprague-Dawley rats and grown to confluence on membrane filters. Mostof the basal short-circuit current(Isc; 60%) wasinhibited by amiloride (IC50 0.96 µM) or benzamil (IC50 0.5 µM).Basolateral addition of terbutaline (2 µM) produced a rapid decreasein Isc, followed by a slow recovery back to its initial amplitude. WhenCl was replaced withmethanesulfonic acid, the basalIsc was reduced and the response to terbutaline was inhibited. In permeabilized monolayer experiments, both terbutaline and amiloride produced sustained decreases in current. The current-voltage relationship of the terbutaline-sensitive current had a reversal potential of28 mV. Increasing Cl concentration in thebasolateral solution shifted the reversal potential to more depolarizedvoltages. These results were consistent with the existence of aterbutaline-activated Cl conductance in the apicalmembrane. Terbutaline did not increase the amiloride-sensitiveNa+ conductance. We conclude that -adrenergicstimulation of adult alveolar epithelial cells results in an increasein apical Cl permeability and thatamiloride-sensitive Na+ channels are not directly affectedby this stimulation.

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14.
Insulin stimulates K+ uptake andNa+ efflux via the Na+-K+ pump inkidney, skeletal muscle, and brain. The mechanism of insulin action inthese tissues differs, in part, because of differences in the isoformcomplement of the catalytic -subunit of theNa+-K+ pump. To analyze specifically the effectof insulin on the 1-isoform of the pump, we have studiedhuman embryonic kidney (HEK)-293 cells stably transfected with the ratNa+-K+ pump 1-isoform tagged onits first exofacial loop with a hemagglutinin (HA) epitope. The plasmamembrane content of 1-subunits was quantitated bybinding a specific HA antibody to intact cells. Insulin rapidly increased the number of 1-subunits at the cell surface.This gain was sensitive to the phosphatidylinositol (PI) 3-kinaseinhibitor wortmannin and to the protein kinase C (PKC) inhibitorbisindolylmaleimide. Furthermore, the insulin-stimulated gain insurface -subunits correlated with an increase in the binding of anantibody that recognizes only the nonphosphorylated form of1 (at serine-18). These results suggest that insulinregulates the Na+-K+ pump in HEK-293 cells, atleast in part, by decreasing serine phosphorylation and increasingplasma membrane content of 1-subunits via a signalingpathway involving PI 3-kinase and PKC.

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15.
Our group recentlycloned the electrogenicNa+-HCO3cotransporter (NBC) from salamander kidney and later from mammaliankidney. Here we report cloning an NBC isoform (hhNBC) from a humanheart cDNA library. hhNBC is identical to human renal NBC (hkNBC),except for the amino terminus, where the first 85 amino acids in hhNBCreplace the first 41 amino acids of hkNBC. About 50% of the amino acidresidues in this unique amino terminus are charged, compared with~22% for the corresponding 41 residues in hkNBC. Northern blotanalysis, with the use of the unique 5' fragment of hhNBC as aprobe, shows strong expression in pancreas and expression in heart andbrain, although at much lower levels. InXenopus oocytes expressing hhNBC,adding 1.5% CO2/10 mMHCO3 hyperpolarizes the membrane andcauses a rapid fall in intracellular pH(pHi), followed by apHi recovery. Subsequent removalof Na+ causes a depolarization anda reduced rate of pHi recovery.Removal of Cl from the bathdoes not affect the pHi recovery.The stilbene derivative DIDS (200 µM) greatly reduces thehyperpolarization caused by addingCO2/HCO3.In oocytes expressing hkNBC, the effects of addingCO2/HCO3and then removing Na+ were similarto those observed in oocytes expressing hhNBC. We conclude that hhNBCis an electrogenicNa+-HCO3cotransporter and that hkNBC is also electrogenic.  相似文献   

16.
Bumetanide blocks CFTR GCl in the native sweat duct   总被引:1,自引:0,他引:1  
Bumetanide is wellknown for its ability to inhibit the nonconductiveNa+-K+-2Clcotransporter. We were surprised in preliminary studies to find thatbumetanide in the contraluminal bath also inhibited NaCl absorption inthe human sweat duct, which is apparently poor in cotransporteractivity. Inhibition was accompanied by a marked decrease in thetransepithelial electrical conductance. Because the cystic fibrosistransmembrane conductance regulator (CFTR) Cl channel is richlyexpressed in the sweat duct, we asked whether bumetanide acts byblocking this anion channel. We found that bumetanide1) significantly increased wholecell input impedance, 2)hyperpolarized transepithelial and basolateral membrane potentials, 3) depolarized apical membranepotential, 4) increased the ratio ofapical-to-basolateral membrane resistance, and5) decreased transepithelialCl conductance(GCl).These results indicate that bumetanide inhibits CFTRGClin both cell membranes of this epithelium. We excluded bumetanideinterference with the protein kinase A phosphorylation activationprocess by "irreversibly" phosphorylating CFTR [by usingadenosine5'-O-(3-thiotriphosphate) in thepresence of a phosphatase inhibition cocktail] before bumetanideapplication. We then activated CFTRGClby adding 5 mM ATP. Bumetanide in the cytoplasmic bath(103 M) inhibited ~71%of this ATP-activated CFTRGCl,indicating possible direct inhibition of CFTRGCl.We conclude that bumetanide inhibits CFTRGClin apical and basolateral membranes independent of phosphorylation. Theresults also suggest that>105 M bumetanide cannotbe used to specifically block theNa+-K+-2Cl cotransporter.

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17.
Pancreatic dysfunction in patients with cystic fibrosis (CF) isfelt to result primarily from impairment of ductalHCO3 secretion. We provide molecularevidence for the expression of NBC-1, an electrogenicNa+-HCO3cotransporter (NBC) in cultured human pancreatic ductcells exhibiting physiological features prototypical of CF ductfragments (CFPAC-1 cells) or normal duct fragments [CAPAN-1 cellsand CFPAC-1 cells transfected with wild-type CF transmembraneconductance regulator (CFTR)]. We further demonstrate that1)HCO3 uptake across the basolateralmembranes of pancreatic duct cells is mediated via NBC and2) cAMP potentiates NBC activitythrough activation of CFTR-mediatedCl secretion. We proposethat the defect in agonist-stimulated ductal HCO3 secretion in patients with CF ispredominantly due to decreased NBC-drivenHCO3 entry at the basolateralmembrane, secondary to the lack of sufficient electrogenic drivingforce in the absence of functional CFTR.

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18.
Serous cells secreteCl and HCO3 and play an importantrole in airway function. Recent studies suggest that aCl/HCO3 anion exchanger (AE) maycontribute to Cl secretion by airway epithelial cells.However, the molecular identity, the cellular location, and thecontribution of AEs to Cl secretion in serous epithelialcells in tracheal submucosal glands are unknown. The goal of thepresent study was to determine the molecular identity, the cellularlocation, and the role of AEs in the function of serous epithelialcells. To this end, Calu-3 cells, a human airway cell line with aserous-cell phenotype, were studied by RT-PCR, immunoblot, andelectrophysiological analysis to examine the role of AEs inCl secretion. In addition, the subcellular location of AEproteins was examined by immunofluorescence microscopy. Calu-3 cellsexpressed mRNA and protein for AE2 as determined by RT-PCR and Westernblot analysis, respectively. Immunofluorescence microscopy identified AE2 in the basolateral membrane of Calu-3 cells in culture and rattracheal serous cells in situ. InCl/HCO3/Na+-containingmedia, the 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate(CPT-cAMP)-stimulated short-circuit anion current (Isc) was reduced by basolateral but not byapical application of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid(50 µM) and 4,4'-dinitrostilbene-2,2'-disulfonic acid [DNDS (500 µM)], inhibitors of AEs. In the absence of Na+ in thebath solutions, to eliminate the contributions of the Na+/HCO3 andNa+/K+/2Cl cotransporters toIsc, CPT-cAMP stimulated a small DNDS-sensitive Isc. Taken together with previous studies, theseobservations suggest that a small component of cAMP-stimulatedIsc across serous cells may be referable toCl secretion and that uptake of Cl acrossthe basolateral membrane may be mediated by AE2.

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19.
Ca2+-mediated agonists,including UTP, are being developed for therapeutic use in cysticfibrosis (CF) based on their ability to modulate alternativeCl conductances. As CF isalso characterized by hyperabsorption ofNa+, we determined the effect ofmucosal UTP on transepithelial Na+transport in primary cultures of human bronchial epithelia (HBE). Insymmetrical NaCl, UTP induced an initial increase in short-circuit current (Isc)followed by a sustained inhibition. To differentiate between effects onNa+ absorption andCl secretion,Isc was measuredin the absence of mucosal and serosal Cl(INa). Again,mucosal UTP induced an initial increase and then a sustained decreasethat reduced amiloride-sensitiveINa by 73%. TheCa2+-dependent agonists histamine,bradykinin, serosal UTP, and thapsigargin similarly induced sustainedinhibition (62-84%) ofINa. Mucosal UTPinduced similar sustained inhibition (half-maximal inhibitory concentration 296 nM) ofINa in primarycultures of human CF airway homozygous for the F508 mutation.BAPTA-AM blunted UTP-dependent inhibition ofINa, butinhibitors of protein kinase C (PKC) and phospholipaseA2 had no effect. Indeed, directactivation of PKC by phorbol 12-myristate 13-acetate failed to inhibitNa+ absorption. Apyrase, a tri-and diphosphatase, did not reverse inhibitory effects of UTP onINa, suggesting along-term inhibitory effect of UTP that is independent of receptoroccupancy. After establishment of a mucosa-to-serosaK+ concentration gradient andpermeabilization of the mucosal membrane with nystatin, mucosal UTPinduced an initial increase in K+current followed by a sustained inhibition. We conclude that increasingcellular Ca2+ induces a long-terminhibition of transepithelial Na+transport across normal and CF HBE at least partly due todownregulation of a basolateral membraneK+ conductance. Thus UTP may havea dual therapeutic effect in CF airway:1) stimulation of aCl secretory response and2) inhibition ofNa+ transport.  相似文献   

20.
Investigation of the role ofindividual protein kinase C (PKC) isozymes in the regulation ofNa+ channels has been largely limited by the lack ofisozyme-selective modulators. Here we used a novel peptide-specificactivator (V1-7) of PKC and other peptide isozyme-specificinhibitors in addition to the general PKC activator phorbol12-myristate 13-acetate (PMA) to dissect the role of individual PKCs inthe regulation of the human cardiac Na+ channel hH1,heterologously expressed in Xenopus oocytes. Peptides wereinjected individually or in combination into the oocyte. Whole cellNa+ current (INa) was recorded usingtwo-electrode voltage clamp. V1-7 (100 nM) and PMA (100 nM)inhibited INa by 31 ± 5% and 44 ± 8% (at 20 mV), respectively. These effects were not seen with thescrambled peptide for V1-7 (100 nM) or the PMA analog4-phorbol 12,13-didecanoate (100 nM). However, V1-7-and PMA-induced INa inhibition was abolished byV1-2, a peptide-specific antagonist of PKC. Furthermore,PMA-induced INa inhibition was not altered by100 nM peptide-specific inhibitors for -, -, -, or PKC. PMAand V1-7 induced translocation of PKC from soluble toparticulate fraction in Xenopus oocytes. This translocationwas antagonized by V1-2. In native rat ventricular myocytes,PMA and V1-7 also inhibited INa; thisinhibition was antagonized by V1-2. In conclusion, the resultsprovide evidence for selective regulation of cardiac Na+channels by PKC isozyme.

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