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.

     

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.     

Apical and basolateral CO2-HCO-3 permeability in cultured bovine corneal endothelial cells

Corneal endothelial function is dependent onHCO₃ transport. However, the relativeHCO₃ permeabilities of the apical andbasolateral membranes are unknown. Using changes in intracellular pHsecondary to removingCO₂-HCO₃ (at constant pH) or removing HCO₃alone (at constant CO₂) fromapical or basolateral compartments, we determined the relative apicaland basolateral HCO₃ permeabilities and their dependencies on Na⁺ andCl. Removal ofCO₂-HCO₃from the apical side caused a steady-state alkalinization (+0.08 pHunits), and removal from the basolateral side caused an acidification(0.05 pH units). Removal ofHCO₃ at constantCO₂ indicated that the basolateralHCO₃ fluxes were about three to fourtimes the apical fluxes. Reducing perfusateNa⁺ concentration to 10 mM had noeffect on apical flux but slowed basolateralHCO₃ flux by one-half. In the absence of Cl, there was anapparent increase in apical HCO₃ fluxunder constant-pH conditions; however, no net change could be measuredunder constant-CO₂ conditions.Basolateral flux was slowed ~30% in the absence ofCl, but the net flux wasunchanged. The steady-state alkalinization after removal ofCO₂-HCO₃apically suggests that CO₂diffusion may contribute to apicalHCO₃ flux through the action of amembrane-associated carbonic anhydrase. Indeed, apicalCO₂ fluxes were inhibited by theextracellular carbonic anhydrase inhibitor benzolamide and partiallyrestored by exogenous carbonic anhydrase. The presence ofmembrane-bound carbonic anhydrase (CAIV) was confirmed byimmunoblotting. We conclude that theNa⁺-dependent basolateralHCO₃ permeability is consistent withNa⁺-nHCO₃cotransport. Changes inHCO₃ flux in the absence ofCl are most likely due toNa⁺-nHCO₃cotransport-induced membrane potential changes that cannot bedissipated. Apical HCO₃ permeabilityis relatively low, but may be augmented byCO₂ diffusion in conjunction witha CAIV.

     

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.

     

Expression of nucleotide-regulated Cl(-) currents in CF and normal mouse tracheal epithelial cell lines

The dominant routefor Cl secretion in mouse tracheal epithelium is viaCl channels different from the cystic fibrosis (CF)transmembrane conductance regulator (CFTR), the channel that isdefective in CF. It has been proposed that the use of purinergicagonists to activate these alternative channels in human airways may bebeneficial in CF. In the present study, two conditionally immortalepithelial cell lines were established from the tracheae of micepossessing the tsA58 T antigen gene, one of which [MTE18-(/)] washomozygous for a knockout of CFTR and the other [MTE7b-(+/)]heterozygous for CFTR expression. In Ussing chamber studies, amiloride(10⁴ M) and a cocktail of cAMP-activating agents(forskolin, IBMX, and dibutyryl cAMP) resulted in small changes in theshort-circuit current (I_sc) and resistance ofboth cell lines, with larger increases in I_scbeing elicited by ionomycin (10⁶ M). Both cell linesexpressed P₂Y₂ receptors and responded to thepurinergic agonists ATP, UTP, and 5'-adenylylimidodiphosphate (10⁴ M) with an increase in I_sc.This response could be inhibited by DIDS and was abolished in thepresence of Cl-free Ringer solution. Reducing the mucosalCl concentration increased the response to UTP of bothcell lines, with a significantly greater increase in MTE18-(/)cells. Pretreatment of these cells with thapsigargin caused a directincrease in I_sc and inhibited the response toUTP. These data suggest that both cell lines expresspurinergic-regulated Cl currents and may prove valuabletools in studying the properties of this pathway.

     

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.

     

AVP V1 receptor-mediated decrease in Cl- efflux and increase in dark cell number in choroid plexus epithelium

The cerebrospinalfluid (CSF)-generating choroid plexus (CP) has manyV₁ binding sites for argininevasopressin (AVP). AVP decreases CSF formation rate and choroidal bloodflow, but little is known about how AVP alters ion transport across theblood-CSF barrier. Adult rat lateral ventricle CP was loaded with³⁶Cl,exposed to AVP for 20 min, and then placed in isotope-free artificial CSF to measure release of³⁶Cl.Effect of AVP at 10¹² to10⁷ M on theCl efflux rate coefficient(in s¹) was quantified.Maximal inhibition (by 20%) ofCl extrusion at10⁹ M AVP was prevented bythe V₁ receptor antagonist[-mercapto-,-cyclopentamethyleneproprionyl¹,O-Me-Tyr²,Arg⁸]vasopressin.AVP also increased by more than twofold the number of dark and possiblydehydrated but otherwise morphologically normal choroid epithelialcells in adult CP. The V₁ receptorantagonist prevented this AVP-induced increment in dark cell frequency.In infant rats (1 wk) with incomplete CSF secretory ability,10⁹ M AVP altered neitherCl efflux nor dark cellfrequency. The ability of AVP to elicit functional and structuralchanges in adult, but not infant, CP epithelium is discussed in regardto ion transport, CSF secretion, intracranial pressure, and hydrocephalus.

     

Functional and molecular characterization of an anion exchanger in airway serous epithelial cells

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 (I_sc) 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 toI_sc, CPT-cAMP stimulated a small DNDS-sensitive I_sc. Taken together with previous studies, theseobservations suggest that a small component of cAMP-stimulatedI_sc across serous cells may be referable toCl secretion and that uptake of Cl acrossthe basolateral membrane may be mediated by AE2.

     

Similarity of A(3)-adenosine and swelling-activated Cl(-) channels in nonpigmented ciliary epithelial cells

Chloride release from nonpigmented ciliary epithelial (NPE)cells is a final step in forming aqueous humor, and adenosine stimulates Cl transport by these cells. Whole cell patchclamping of cultured human NPE cells indicated that theA₃-selective agonist1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl--D-ribofuranuronamide (IB-MECA) stimulated currents (I_IB-MECA) by~90% at +80 mV. Partial replacement of external Clwith aspartate reduced outward currents and shifted the reversal potential (V_rev) from 23 ± 2 mV to0.0 ± 0.7 mV. Nitrate substitution had little effect. Perfusionwith the Cl channel blockers5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acidinhibited the currents. Partial Cl replacement withaspartate and NO₃, and perfusion with NPPB, hadsimilar effects on the swelling-activated whole cell currents(I_Swell). Partial cyclamate substitution for external Cl inhibited inward and outward currents of bothI_IB-MECA and I_Swell. Bothsets of currents also showed outward rectification and inactivation atlarge depolarizing potentials. The results are consistent with theconcept that A₃-subtype adenosine agonists and swellingactivate a common population of Cl channels.

     

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.

     

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.

     

TGF-alpha reduces bradykinin-stimulated ion transport and prostaglandin release in human colonic epithelial cells

The effect of chronic exposure to transforming growth factor-(TGF-) on bradykinin-stimulated acute prostanoid production and ionsecretion in monolayers of HCA-7 colony 29 colonic epithelial cells hasbeen studied. Monolayers synthesized prostaglandinE₂ (PGE₂) at a basal rate of 2.10 ± 0.31 pg · monolayer¹ · min¹over 24 h. Bradykinin(10⁸-10⁵M) dose dependently increased acutePGE₂ release by three orders ofmagnitude. This was associated with a rise in cAMP from 1.60 ± 0.14 to 2.90 ± 0.1 pmol/monolayer (P < 0.02) and a dose-dependent increase in short-circuit current (SCC).When monolayers were primed by a 24-h exposure to TGF-, basalPGE₂ release rose to 6.31 ± 0.38 pg · monolayer¹ · min¹(TGF- concn 10 ng/ml; P = 0.001).However, the stimulation of acute prostaglandin release, intracellularcAMP, and increased SCC by bradykinin was significantly reduced bypreincubation with TGF-. Priming withPGE₂(10⁸-10⁶M) over 24 h mimicked the effect of TGF- on bradykinin-induced changes in cAMP and SCC. These data suggest that enhanced chronic release of prostaglandins in response to stimulation with TGF- maydownregulate acute responses to bradykinin. In vivo, TGF- could havean important modulatory function in regulating secretion underinflammatory conditions.     

Nerve growth factor regulates HCO-3 absorption in thick ascending limb: modifying effects of vasopressin

Growth factorsstimulateNa⁺/H⁺exchange activity in many cell types but their effects on acidsecretion via this mechanism in renal tubules are poorly understood. Weexamined the regulation of HCO₃absorption by nerve growth factor (NGF) in the rat medullary thickascending limb (MTAL), which absorbs HCO₃via apical membraneNa⁺/H⁺exchange. MTAL were perfused in vitro with 25 mMHCO₃ solutions (pH 7.4; 290 mosmol/kgH₂O). Addition of 0.7 nMNGF to the bath decreased HCO₃absorption from 13.1 ± 1.1 to 9.6 ± 0.8 pmol · min¹ · mm¹(P < 0.001). In contrast, with10¹⁰ M arginine vasopressin(AVP) in the bath, addition of NGF to the bath increasedHCO₃ absorption from 8.0 ± 1.6 to12.5 ± 1.3 pmol · min¹ · mm¹(P < 0.01). Both effects of NGF wereblocked by genistein, consistent with the involvement of tyrosinekinase pathways. However, the AVP-dependent stimulation requiredactivation of protein kinase C (PKC), whereas the inhibition was PKCindependent, indicating that the NGF-induced signaling pathways leadingto inhibition and stimulation of HCO₃absorption are distinct. Hypertonicity blocked the inhibition but notthe AVP-dependent stimulation, suggesting that hypertonicity and NGFmay inhibit HCO₃ absorption via acommon mechanism. These data demonstrate that NGF inhibitsHCO₃ absorption in the MTAL underbasal conditions but stimulates HCO₃ absorption in the presence of AVP, effects that are mediated through distinct signal transduction pathways. They also show that AVP is acritical determinant of the response of the MTAL to growth factorstimulation and suggest that NGF can either inhibit or stimulateapical Na⁺/H⁺ exchange activitydepending on its interactions with other regulatory factors. Locallyproduced growth factors such as NGF may play a role in regulating renaltubule HCO₃ absorption.

     

Human cytomegalovirus infection stimulates Cl-/HCO-3 exchanger activity in human fibroblasts

The effects ofhuman cytomegalovirus (HCMV) infection onCl/HCO₃exchanger activity in human lung fibroblasts (MRC-5 cells) were studiedusing fluorescent, ion-sensitive dyes. The intracellular pH(pH_i) of mock- and HCMV-infectedcells bathed in a solution containing 5%CO₂-25 mMHCO₃ were nearly the same. However,replacement of external Clwith gluconate caused anH₂DIDS-inhibitable (100 µM)increase in the pH_i ofHCMV-infected cells but not in mock-infected cells. Continuous exposureto hyperosmotic external media containing CO₂/HCO₃caused the pH_i of both cell typesto increase. The pH_i remainedelevated in mock-infected cells. However, in HCMV-infected cells, thepH_i peaked and then recoveredtoward control values. This pH_irecovery phase was completely blocked by 100 µMH₂DIDS. In the presence ofCO₂/HCO₃, there was an H₂DIDS-sensitivecomponent of net Cl efflux(external Cl wassubstituted with gluconate) that was less in mock- than in HCMV-infected cells. When nitrate was substituted for external Cl (in the nominal absenceofCO₂/HCO₃),the H₂DIDS-sensitive netCl efflux was much greaterfrom HCMV- than from mock-infected cells. In mock-infected cells,H₂DIDS-sensitive, netCl efflux decreased aspH_i increased, whereas forHCMV-infected cells, efflux increased aspH_i increased. All these resultsare consistent with an HCMV-induced enhancement ofCl/HCO₃exchanger activity.

     

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.

     

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.

     

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.

     

Long-wavelength iodide-sensitive fluorescent indicators for measurement of functional CFTR expression in cells

Limitations of available indicators [such as6-methoxy-N-(3-sulfopropyl)quinolinium(SPQ)] for measurement of intracellular Cl are their relatively dimfluorescence and need for ultraviolet excitation. A series oflong-wavelength polar fluorophores was screened to identify compoundswith Cl and/orI sensitivity, brightfluorescence, low toxicity, uniform loading of cytoplasm with minimalleakage, and chemical stability in cells. The best compound found was7-(-D-ribofuranosylamino)-pyrido[2,1-h]-pteridin-11-ium-5-olate (LZQ). LZQ is brightly fluorescent with excitation andemission maxima at 400-470 and 490-560 nm, molar extinction11,100 M¹ · cm¹(424 nm), and quantum yield 0.53. LZQ fluorescence is quenched byI by a collisionalmechanism (Stern-Volmer constant 60 M¹) and is not affectedby other halides, nitrate, cations, or pH changes (pH 5-8). AfterLZQ loading into cytoplasm by hypotonic shock or overnight incubation,LZQ remained trapped in cells (leakage <3%/h). LZQ stained cytoplasmuniformly, remained chemically inert, did not bind to cytoplasmiccomponents, and was photobleached by <1% during 1 h of continuousillumination. Cytoplasmic LZQ fluorescence was quenched selectively byI (50% quenching at 38 mMI). LZQ was used tomeasure forskolin-stimulatedI/ClandI/NO₃exchange in cystic fibrosis transmembrane conductance regulator(CFTR)-expressing cell lines by fluorescence microscopy and microplatereader instrumentation using 96-well plates. The substantially improvedoptical and cellular properties of LZQ over existing indicators shouldpermit the quantitative analysis of CFTR function in gene deliverytrials and high-throughput screening of compounds for correction of thecystic fibrosis phenotype.

     

Contrasting effects of NO and peroxynitrites on HSP70 expression and apoptosis in human monocytes

The free radicals nitric oxide(·NO) and superoxide (O₂·) react to formperoxynitrite (ONOO), a highly toxic oxidant species. Inthis study we investigated the respective effects of NO andONOO in monocytes from healthy human donors. Purifiedmonocytes were incubated for 6 or 16 h with a pure NO donor(S-nitroso-N-acetyl-DL-penicillamine, 0-2 mM), an ·NO/ONOO donor(3-morpholinosydnonimine chlorhydrate, 0-2 mM) with and withoutsuperoxide dismutase (200 IU/ml), or pure ONOO. Weprovide evidence that 3-morpholinosydnonimine chlorhydrate alonerepresents a strong stress to human monocytes leading to adose-dependent increase in heat shock protein-70 (HSP70) expression, mitochondrial membrane depolarization, and cell death by apoptosis andnecrosis. These phenomena were abolished by superoxide dismutase, suggesting that ONOO, but not ·NO, was responsible forthe observed effects. This observation was further strengthened by theabsence of a stress response in cells exposed toS-nitroso-N-acetyl-DL-penicillamine. Conversely, exposure of cells to ONOO alone also inducedmitochondrial membrane depolarization and cell death by apoptosis andnecrosis. Thus ONOO formation may well explain the toxiceffect generally attributed to ·NO.

     

Alterations in AMP deaminase activity and kinetics in skeletal muscle of creatine kinase-deficient mice

Alterations in the competency of the creatine kinase systemelicit numerous structural and metabolic compensations, including changes in purine nucleotide metabolism. We evaluated molecular andkinetic changes in AMP deaminase from skeletal muscles of micedeficient in either cytosolic creatine kinase alone(M-CK^/) or alsodeficient in mitochondrial creatine kinase(CK^/) comparedwith wild type. We found that predominantly fast-twitch muscle, but notslow-twitch muscle, from bothM-CK^/ andCK^/ mice had muchlower AMP deaminase; the quantity of AMP deaminase detected by Westernblot was correspondingly lower, whereas AMP deaminase-1(AMPD1) gene expressionwas unchanged. Kinetic analysis of AMP deaminase from mixed musclerevealed negative cooperativity that was significantly greater increatine kinase deficiencies. Treatment of AMP deaminase with acidphosphatase abolished negative cooperative behavior, indicating that aphosphorylation-dephosphorylation cycle may be important in theregulation of AMP deaminase.