Osmotic regulation of intestinal epithelial Na+-K+-Cl- cotransport: role of Cl- and F-actin

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.

     

Astrocytes from Na(+)-K(+)-Cl(-) cotransporter-null mice exhibit absence of swelling and decrease in EAA release

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.

     

Synthesis and characterization of dual-wavelength Cl--sensitive fluorescent indicators for ratio imaging

The fluorescence of quinolinium-basedCl indicators such as6-methoxy-N-(3-sulfopropyl)quinolinium(SPQ) is quenched by Cl bya collisional mechanism without change in spectral shape. A series of"chimeric" dual-wavelengthCl indicators weresynthesized by conjugatingCl-sensitive and-insensitive chromophores with spacers. The SPQ chromophore(N-substituted 6-methoxyquinolinium; MQ) was selected as theCl-sensitive moiety[excitation wavelength(_ex) 350 nm, emission wavelength (_em) 450 nm]. N-substituted 6-aminoquinolinium (AQ) waschosen as theCl-insensitive moietybecause of its different spectral characteristics (_ex 380 nm,_em 546 nm), insensitivity toCl, positive charge (tominimize quenching by chromophore stacking/electron transfer), andreducibility (for noninvasive cell loading). The dual-wavelengthindicators were stable and nontoxic in cells and were distributeduniformly in cytoplasm, with occasional staining of the nucleus. Thebrightest and mostCl-sensitive indicatorswere -MQ-'-dimethyl-AQ-xylene dichloride andtrans-1,2-bis(4-[1-'-MQ-1'-'-dimethyl-AQ-xylyl]-pyridinium)ethylene (bis-DMXPQ). At 365-nm excitation, emission maxima were at 450 nm(Cl sensitive; Stern-Volmerconstants 82 and 98 M¹)and 565 nm (Clinsensitive). Cystic fibrosis transmembrane conductanceregulator-expressing Swiss 3T3 fibroblasts were labeled with bis-DMXPQby hypotonic shock or were labeled with its uncharged reduced form(octahydro-bis-DMXPQ) by brief incubation (20 µM, 10 min). Changes inCl concentration inresponse to Cl/nitrateexchange were recorded by emission ratio imaging (450/565 nm) at 365-nmexcitation wavelength. These results establish a first-generation setof chimeric bisquinoliniumCl indicators forratiometric measurement ofCl concentration.     

CFTR induces the expression of DRA along with Cl(-)/HCO(3)(-) exchange activity in tracheal epithelial cells

Thickening of airway mucus and lungdysfunction in cystic fibrosis (CF) results, at least in part, fromabnormal secretion of Cl and HCO₃across the tracheal epithelium. The mechanism of the defect in HCO₃ secretion is ill defined; however, a lack ofapical Cl/HCO₃ exchange may exist inCF. To test this hypothesis, we examined the expression ofCl/HCO₃ exchangers in trachealepithelial cells exhibiting physiological features prototypical ofcystic fibrosis [CFT-1 cells, lacking a functional cystic fibrosistransmembrane conductance regulator (CFTR)] or normal trachea (CFT-1cells transfected with functional wild-type CFTR, termed CFT-WT). Cellswere grown on coverslips and were loaded with the pH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, andintracellular pH was monitored. Cl/HCO₃exchange activity increased by ~300% in cells transfected with functional CFTR, with activities increasing from 0.034 pH/min in CFT-1cells to 0.11 in CFT-WT cells (P < 0.001, n = 8). This activity was significantly inhibited byDIDS. The mRNA expression of the ubiquitous basolateral AE-2Cl/HCO₃ exchanger remained unchanged.However, mRNA encoding DRA, recently shown to be aCl/HCO₃ exchanger (Melvin JE, Park K,Richardson L, Schultheis PJ, and Shull GE. J Biol Chem 274:22855-22861, 1999.) was abundantly expressed in cells expressingfunctional CFTR but not in cells that lacked CFTR or that expressedmutant CFTR. In conclusion, CFTR induces the mRNA expression of"downregulated in adenoma" (DRA) and, as a result, upregulates theapical Cl/HCO₃ exchanger activity intracheal cells. We propose that the tracheal HCO₃secretion defect in patients with CF is partly due to thedownregulation of the apical Cl/HCO₃exchange activity mediated by DRA.

     

ANG II controls Na+-K+(NH+4)-2Cl- cotransport via 20-HETE and PKC in medullary thick ascending limb

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.

     

Distinct Ca2+- and cAMP-dependent anion conductances in the apical membrane of polarized T84 cells

Monolayers of the human colonic epithelial cell line T84 exhibitelectrogenic Cl secretionin response to the Ca²⁺ agonistthapsigargin and to the cAMP agonist forskolin. To evaluate directlythe regulation of apical Clconductance by these two agonists, we have utilized amphotericin B topermeabilize selectively the basolateral membranes of T84 cellmonolayers. We find that apical anion conductance is stimulated by bothforskolin and thapsigargin but that these conductances aredifferentially sensitive to the anion channel blocker DIDS. DIDSinhibits thapsigargin-stimulated responses completely but forskolinresponses only partially. Furthermore, the apical membrane anionconductances elicited by these two agonists differ in anion selectivity(for thapsigargin, I > Cl; for forskolin,Cl > I). However, theDIDS-sensitive component of the forskolin-induced conductance responseexhibits anion selectivity similar to that induced by thapsigargin(I > Cl). Thusforskolin-induced apical anion conductance comprises at least twocomponents, one of which has features in common with that elicited bythapsigargin.

     

PKA and arachidonic acid activation of human recombinant ClC-2 chloride channels

An HEK-293 cell line stably expressing the humanrecombinant ClC-2 Cl channel was used in patch-clampstudies to study its regulation. The relative permeabilityP_x/P_Cl calculated fromreversal potentials was I > Cl = NO₃ = SCNBr. Theabsolute permeability calculated from conductance ratios wasCl = Br = NO₃  SCN > I. The channel was activatedby cAMP-dependent protein kinase (PKA), reduced extracellular pH, oleicacid (C:18 cis9), elaidic acid (C:18trans9), arachidonic acid (AA; C:20cis5,8,11,14), and by inhibitors of AA metabolism,5,8,11,14-eicosatetraynoic acid (ETYA; C:20trans5,8,11,14),-methyl-4-(2-methylpropyl)benzeneacetic acid (ibuprofen), and2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2Cl channels were activated by a combination of forskolinplus IBMX and were inhibited by the cell-permeant myristoylated PKAinhibitor (mPKI). Channel activation by reduction of bath pH wasincreased by PKA and prevented by mPKI. AA activation of the ClC-2Cl channel was not inhibited by mPKI or staurosporine andwas therefore independent of PKA or protein kinase C activation.

     

CFTR upregulates the expression of the basolateral Na+-K+-2Cl- cotransporter in cultured pancreatic duct cells

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.

     

Exocytosis is not involved in activation of Cl- secretion via CFTR in Calu-3 airway epithelial cells

Cystic fibrosis iscaused by mutations in the cystic fibrosis transmembrane conductanceregulator (CFTR) Clchannel, which mediates transepithelialCl transport in a varietyof epithelia, including airway, intestine, pancreas, and sweat duct. Insome but not all epithelial cells, cAMP stimulatesCl secretion in part byincreasing the number of CFTRCl channels in the apicalplasma membrane. Because the mechanism whereby cAMP stimulates CFTRCl secretion is cell-typespecific, our goal was to determine whether cAMP elevates CFTR-mediatedCl secretion across serousairway epithelial cells by stimulating the insertion of CFTRCl channels from anintracellular pool into the apical plasma membrane. To this end westudied Calu-3 cells, a human airway cell line with a serous cellphenotype. Serous cells in human airways, such as Calu-3 cells, expresshigh levels of CFTR, secrete antibiotic-rich fluid, and play a criticalrole in airway function. Moreover, dysregulation of CFTR-mediatedCl secretion in serouscells is thought to contribute to the pathophysiology of cysticfibrosis lung disease. We report that cAMP activation of CFTR-mediatedCl secretion across humanserous cells involves stimulation of CFTR channels present in theapical plasma membrane and does not involve the recruitment of CFTRfrom an intracellular pool to the apical plasma membrane.

     

Potentiation of nitric oxide-induced apoptosis in p53-/- vascular smooth muscle cells

The functionalrole of p53 in nitric oxide (NO)-mediated vascular smooth muscle cell(VSMC) apoptosis remains unknown. In this study, VSMC fromp53^/ and p53^+/+ murine aortas were exposedto exogenous or endogenous sources of NO. Unexpectedly,p53^/ VSMC were much more sensitive to theproapoptotic effects of NO than were p53^+/+ VSMC.Furthermore, this paradox appeared to be specific to NO, because otherproapoptotic agents did not demonstrate this differential effect onp53^/ cells. NO-induced apoptosis inp53^/ VSMC occurred independently of cGMP generation.However, mitogen-activated protein kinase (MAPK) pathways appeared toplay a significant role. Treatment of the p53^/ VSMCwith S-nitroso-N-acetylpenicillamine resulted ina marked activation of p38 MAPK and, to a lesser extent, of c-JunNH₂-terminal kinase, mitogen-activated protein kinasekinase (MEK) 1/2, and p42/44 (extracellular signal-regulated kinase,ERK). Furthermore, basal activity of the MEK-p42/44 (ERK)pathway was increased in the p53^+/+ VSMC. Inhibition of p38MAPK with SB-203580 or of MEK1/2 with PD-98059 blocked NO-inducedapoptosis. Therefore, p53 may protect VSMC against NO-mediatedapoptosis, in part, through differential regulation of MAPK pathways.

     

CFTR drives Na+-nHCO-3 cotransport in pancreatic duct cells: a basis for defective HCO-3 secretion in CF

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.

     

Na+-K+-Cl- cotransport in human fibroblasts is inhibited by cytomegalovirus infection

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.

     

Inhibition of proximal tubular fluid absorption by nitric oxide and atrial natriuretic peptide in rat kidney

Atrial natriuretic factor (ANF) and nitric oxide (NO) stimulateproduction of guanosine 3',5'-cyclic monophosphate (cGMP) and are natriuretic. Split-drop micropuncture was performed on anesthetized rats to determine the effects of ANF and the NO donor sodium nitroprusside (SNP) on proximal tubular fluid absorption rate(J_va). Comparedwith control solutions, SNP(10⁴ M) decreasedJ_va by 23% whenadministered luminally and by 35% when added to the peritubularperfusate. Stimulation of fluid uptake by luminal angiotensin II (ANGII; 10⁹ M) was abolished bySNP (10⁴ and10⁶ M). In proximal tubulesuspensions, ANF (10⁶ M)increased cGMP concentration to 143%, whereas SNP(10⁶,10⁵,10⁴,10³ M) raised cGMP to 231, 594, 687, and 880%, respectively.S-nitroso-N-acetylpenicillamine (SNAP) also raised cGMP concentrations with similar dose-response relations. These studies demonstrate inhibition by luminal and peritubular NO of basal and ANG II-stimulated proximal fluid absorption in vivo. The ability of SNP to inhibit basal fluid uptake whereas ANFonly affected ANG II-stimulated transport may be because of productionof higher concentrations of cGMP by SNP.

     

Identification of an acid-activated Cl- channel from human skeletal muscles

ClC-4 gene was isolated as a putativeCl channel. Due to a lackof functional expression of ClC-4, its physiological role remainsunknown. We isolated a human ClC-4 clone (hClC-4sk) from human skeletalmuscles and stably transfected it to Chinese hamster ovary cells. Wholecell patch-clamp studies showed that the hClC-4sk channel was activatedby external acidic pH and inhibited by DIDS. It passed a strong outwardCl current with apermeability sequence of I > Cl > F. The hClC-4sk hasconsensus sites for phosphorylation by protein kinase A (PKA); however,stimulation of PKA had no effect on the currents. hClC-4sk mRNA wasexpressed in excitable tissues, such as heart, brain, and skeletalmuscle. These functional characteristics of hClC-4sk provide a clue toits physiological role in excitable cells.

     

B16-BL6 melanoma cells release inhibitory factor(s) of active pump activity in isolated lymph vessels

We investigated whethersupernatant cultured with melanoma cell lines B16-BL6 and K1735 or theLewis lung carcinoma cell line (LLC) can regulate lymphatic pumpactivity with bioassay preparations isolated from murine iliac lymphvessels. B16-BL6 and LLC supernatants caused significantdilation of lymph microvessels with cessation of pump activity. B16-BL6supernatant produced dose-related cessation of lymphatic pump activity.There was no significant tachyphylaxis in the supernatant-mediatedinhibitory response of lymphatic pump activity. Pretreatment with3 × 10⁵ MN-nitro-L-arginine methyl ester(L-NAME) or 10⁷ M or 10⁶ Mglibenclamide and 5 × 10⁴ M 5-hydroxydecanoic acidcaused significant reduction of supernatant-mediated inhibitoryresponses. Simultaneous treatment with 10³ ML-arginine and 3 × 10⁵ ML-NAME significantly lessened L-NAME-inducedinhibition of the supernatant-mediated response, suggesting thatendogenous nitric oxide (NO) plays important roles insupernatant-mediated inhibitory responses. Chemical treatment dialyzedsubstances of <1,000 molecular weight (MW), producing completereduction of the supernatant-mediated response. In contrast,pretreatment with heating or digestion with protease had no significanteffect on supernatant-mediated response. These findings suggest thatB16-BL6 cells may release nonpeptide substance(s) of <1,000 MW,resulting in significant cessation of lymphatic pump activity viaproduction and release of endogenous NO and activation of mitochondrialATP-sensitive K⁺ channels.

     

Characterization of CFTR expression and chloride channel activity in human endothelia

The cystic fibrosis transmembrane conductance regulator (CFTR)functions as a low-conductance, cAMP-regulated chloride(Cl) channel in a varietyof cell types, such as exocrine epithelial cells. Our resultsdemonstrate that human primary endothelial cells isolated fromumbilical vein (HUVEC) and lung microvasculature (HLMVEC) also expressCFTR as determined via RT-PCR and immunohistochemical andimmunoprecipitation analyses. Moreover,Cl efflux and whole cellpatch-clamp analyses reveal that HUVEC (n = 6 samples,P < 0.05) and HLMVEC(n = 5 samples,P < 0.05) display cyclicnucleotide-stimulated Cltransport that is inhibited by the CFTR selectiveCl channel blockerglibenclamide but not by the blocker DIDS, indicative of CFTRCl channel activity. Takentogether, these findings demonstrate that human endothelial cellsderived from multiple organ systems express CFTR and that CFTRfunctions as a cyclic nucleotide-regulated Cl channel in human endothelia.

     

Effect of HCO(3)(-) on TPA- and IBMX-induced anion conductances in Necturus gallbladder epithelial cells

Effects of HCO₃ on protein kinase C (PKC)-and protein kinase A (PKA)-induced anion conductances were investigatedin Necturus gallbladder epithelial cells. InHCO₃-free media, activation of PKC via12-O-tetradecanoylphorbol 13-acetate (TPA) depolarizedapical membrane potential (V_a) and decreased fractional apical voltage ratio (F_R). These effects wereblocked by mucosal 5-nitro-2-(3-phenylpropylamino) benzoic acid(NPPB), a Cl channel blocker. In HCO₃media, TPA induced significantly greater changes inV_a and F_R. These effects wereblocked only when NPPB was present in both mucosal and basolateralcompartments. The data suggest that TPA activates NPPB-sensitive apicalCl conductance (g_Cl^a) in theabsence of HCO₃; in its presence, TPA stimulated bothNPPB-sensitive g_Cl^a and basolateralCl conductance (g_Cl^b).Activation of PKA via 3-isobutyl-1-methylxanthine (IBMX) also decreased V_a and F_R; however, thesechanges were not affected by external HCO₃. Weconclude that HCO₃ modulates the effects of PKC ong_Cl^b. In HCO₃ medium, TPAand IBMX also induced an initial transient hyperpolarization andincrease in intracellular pH. Because these changes were independent ofmucosal Na⁺ and Cl, it is suggested that TPAand IBMX induce a transient increase in apical HCO₃ conductance.

     

Cloning and characterization of a human electrogenic Na+-HCO-3 cotransporter isoform (hhNBC)

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(pH_i), followed by apH_i recovery. Subsequent removalof Na⁺ causes a depolarization anda reduced rate of pH_i recovery.Removal of Cl from the bathdoes not affect the pH_i 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.     

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.

     

Peroxynitrite inhibits amiloride-sensitive Na+ currents in Xenopus oocytes expressing alpha beta gamma -rENaC

We examined the effect of peroxynitrite(ONOO) on the cloned ratepithelial Na⁺ channel(-rENaC) expressed in Xenopusoocytes. 3-Morpholinosydnonimine (SIN-1) was used to concurrentlygenerate nitric oxide (· NO) and superoxide(O₂ ·), which react toform ONOO, a species knownto promote protein nitration and oxidation. Under control conditions,oocytes displayed an amiloride-sensitive whole cell conductance of 7.4 ± 2.8 (SE) µS. When incubated at 18°C with SIN-1 (1 mM) for 2 h (final ONOO concentration = 10 µM), the amiloride-sensitive conductance was reduced to0.8 ± 0.5 µS. To evaluate whether the observed inhibition was due to ONOO, as opposedto · NO, we also exposed oocytes to SIN-1 in the presence ofurate (500 µM), a scavenger ofONOO and superoxidedismutase, which scavengesO₂ ·, converting SIN-1from an ONOO to an· NO donor. Under these conditions, conductance values remained at control levels following SIN-1 treatment.Tetranitromethane, an agent that oxidizes sulfhydryl groups at pH6, also inhibited the amiloride-sensitive conductance. These datasuggest that oxidation of critical sulfhydryl groups within rENaC byONOO directly inhibitschannel activity.