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.

     

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.     

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.

     

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.

     

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.     

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.

     

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.

     

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.

     

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.

     

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.

     

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.

     

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.

     

Rabbit conjunctival epithelium transports fluid, and P2Y22 receptor agonists stimulate Cl{-} and fluid secretion

Rabbit conjunctival epithelium exhibits UTP-dependentCl secretion into the tears. We investigated whetherfluid secretion also takes place. Short-circuit current(I_sc) was 14.9 ± 1.4 µA/cm²(n = 16). Four P2Y₂ purinergic receptoragonists [UTP and the novel compounds INS365, INS306, and INS440(Inspire Pharmaceuticals)] added apically (10 µM) resulted intemporary (~30 min) I_sc increases (88%, 66%,57%, and 28%, respectively; n = 4 each). Importantly, the conjunctiva transported fluid from serosa to mucosa at a rate of6.5 ± 0.7 µl · h¹ · cm² (range2.1-15.3, n = 20). Fluid transport was stimulatedby mucosal additions of 10 µM: 1) UTP, from 7.4 ± 2.3 to 10.7 ± 3.3 µl · h¹ · cm²,n = 5; and 2) INS365, from 6.3 ± 1.0 to 9.8 ± 2.5 µl · h¹ · cm²,n = 5. Fluid transport was abolished by 1 mMouabain (n = 5) and was drastically inhibited by 300 µM quinidine (from 6.4 ± 1.2 to 3.6 ± 1.0 µl · h¹ · cm²,n = 4). We conclude that this epithelium secretes fluidactively and that P2Y₂ agonists stimulate bothCl and fluid secretions.

     

Direct inhibitory effect of CCCP on the Cl--H+ symporter of the guinea pig ileal brush-border membrane

The effect of carbonylcyanide-m-chlorophenylhydrazone (CCCP)on Cl uptake across thebrush-border membrane (BBM) was quantified using³⁶Cl and BBM vesicles from guineapig ileum. CCCP inhibited only partially both the pH gradient-activatedCl uptake andCl/Clexchange activities present in these vesicles. In contrast, CCCP had noeffect on the initial (2-30 s) decay rate of an imposed proton gradient, as determined using the pH-sensitive fluorophore pyranine. Taken together, these results strongly indicate that the mainaction of CCCP does not consist of dissipating any imposed pH gradientbut rather in inhibiting directly the pH gradient-activated Cl uptake andCl/Clexchange activities characterizing the intestinal BBM. Because thesetwo activities can be explained in terms of a single (homogeneous) random, nonobligatory two-siteCl-H⁺symporter, in whichCl/Clexchange occurs by counterflow [F. Alvarado and M. Vasseur.Am. J. Physiol. 271 (Cell Physiol. 40): C1612-C1628,1996], we developed a new, more general three-site symport modelthat fully explains the Cluptake inhibitions caused by CCCP. This new model postulates theexistence of a third, allosteric, inhibitory CCCP-binding site separatefrom either of the two substrate-binding sites of theCl-H⁺symporter, the Cl-bindingand the H⁺-binding sites. Finally,we show that, to explain the partial inhibitions observed, it isnecessary to postulate that all the substrate-bound carrier complexes,=C-S, I=C-S, A=C-S, and IA=C-S, where C is carrier, I is inhibitor, Sis substrate, and A is activator, can form and be translocated.

     

Transport of fluid by lens epithelium

We report for the first time that cultured lens epithelial celllayers and rabbit lenses in vitro transport fluid. Layers of the TN4mouse cell line and bovine cell cultures were grown to confluence onpermeable membrane inserts. Fluid movement across cultured layers andexcised rabbit lenses was determined by volume clamp (37°C).Cultured layers transported fluid from their basal to their apicalsides against a pressure head of 3 cmH₂O. Rates were (inµl · h¹ · cm²)3.3 ± 0.3 for TN4 cells (n = 27) and 4.7 ± 1.0 for bovine layers (n = 6). Quinidine, a blocker ofK⁺ channels, andp-chloromercuribenzenesulfonate andHgCl₂, inhibitors of aquaporins,inhibited fluid transport. Rabbit lenses transported fluid from theiranterior to their posterior sides against a2.5-cmH₂O pressure head at 10.3 ± 0.62 µl · h¹ · lens¹(n = 5) and along the same pressurehead at 12.5 ± 1.1 µl · h¹ · lens¹(n = 6). We calculate that this flowcould wash the lens extracellular space by convection about once every2 h and therefore might contribute to lens homeostasis and transparency.     

Cloning, characterization, and functional expression of a CNP receptor regulating CFTR in the shark rectal gland

In the shark, C-type natriuretic peptide (CNP) is the onlycardiac natriuretic hormone identified and is a potent activator ofCl secretion in the rectalgland, an epithelial organ of this species that contains cysticfibrosis transmembrane conductance regulator (CFTR) Clchannels. We have cloned an ancestral CNP receptor (NPR-B) from theshark rectal gland that has an overall amino acid identity to the humanhomologue of 67%. The shark sequence maintains six extracellular Cyspresent in other NPR-B but lacks a glycosylation site and a Glu residuepreviously considered important for CNP binding. When shark NPR-B andhuman CFTR were coexpressed in Xenopus oocytes, CNP increased the cGMP content of oocytes(EC₅₀ 12 nM) and activated CFTRCl channels(EC₅₀ 8 nM). Oocyte cGMP increased36-fold (from 0.11 ± 0.03 to 4.03 ± 0.45 pmol/oocyte) andCl current increased37-fold (from 34 ± 14 to 1,226 ± 151 nA) in thepresence of 50 nM CNP. These findings identify the specific natriureticpeptide receptor responsible forCl secretion in the sharkrectal gland and provide the first evidence for activation of CFTRCl channels by a clonedNPR-B receptor.     

Peroxynitrite causes endothelial cell monolayer barrier dysfunction

Nitric oxide (·NO) attenuates hydrogen peroxide(H₂O₂)-mediated barrier dysfunction in culturedporcine pulmonary artery endothelial cells (PAEC) (Gupta MP, Ober MD,Patterson C, Al-Hassani M, Natarajan V, and Hart, CM. Am JPhysiol Lung Cell Mol Physiol 280: L116-L126, 2001). However,·NO rapidly combines with superoxide (O) to formthe powerful oxidant peroxynitrite (ONOO), which wehypothesized would cause PAEC monolayer barrier dysfunction. To testthis hypothesis, we treated PAEC with ONOO (500 µM) or3-morpholinosydnonimine hydrochloride (SIN-1; 1-500 µM).SIN-1-mediated ONOO formation was confirmed by monitoringthe oxidation of dihydrorhodamine 123 to rhodamine. BothONOO and SIN-1 increased albumin clearance(P < 0.05) in the absence of cytotoxicity and alteredthe architecture of the cytoskeletal proteins actin and -catenin asdetected by immunofluorescent confocal imaging.ONOO-induced barrier dysfunction was partially reversibleand was attenuated by cysteine. Both ONOO and SIN-1nitrated tyrosine residues, including those on -catenin and actin,and oxidized proteins in PAEC. The introduction of actin treated withONOO into PAEC monolayers via liposomes alsoresulted in barrier dysfunction. These results indicate thatONOO directly alters endothelial cytoskeletal proteins,leading to barrier dysfunction.

     

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.

     

Role for anions in pulmonary endothelial permeability

Griffin, M. Pamela. Role for anions in pulmonaryendothelial permeability. J. Appl.Physiol. 83(2): 615-622, 1997.-Adrenergic stimulation reduces albumin permeation across pulmonary artery endothelial monolayers and induces changes in cell morphology that aremediated by Cl flux. Wetested the hypothesis that anion-mediated changes in endothelial cellsresult in changes in endothelial permeability. We measured permeationof radiolabeled albumin across bovine pulmonary arterial endothelialmonolayers when the extracellular anion was Cl,Br,I,F, acetate(Ac), gluconate(G), and propionate(Pr). Permeability toalbumin (P_albumin)was calculated before and after addition of 0.2 mM of thephosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), whichreduces permeability. InCl, theP_albumin was 3.05 ± 0.86 × 10⁶ cm/s andfell by 70% with the addition of IBMX. The initialP_albumin was lowest forPr andAc. InitialP_albumin was higher inBr,I,G, andF than inCl. A permeability ratiowas calculated to examine the IBMX effect. The greatest IBMX effect wasseen when Cl was theextracellular anion, and the order among halide anions wasCl > Br > I > F. Although the level ofextracellular Ca²⁺ concentration([Ca²⁺]_o)varied over a wide range in the anion solutions,[Ca²⁺]_odid not systematically affect endothelial permeability in this system.When Cl was theextracellular anion, varying[Ca²⁺]_ofrom 0.2 to 2.8 mM caused a change in initialP_albumin but no changein the IBMX effect. The anion channel blockers4-acetamido-4-isothiocyanotostilbene-2,2-disulfonic acid(0.25 mM) and anthracene-9-carboxylic acid (0.5 mM) significantly altered initialP_albumin and the IBMXeffect. The anion transport blockers bumetanide (0.2 mM) and furosemide(1 mM) had no such effects. We conclude that extracellular anionsinfluence bovine pulmonary arterial endothelial permeability and thatthe pharmacological profile fits better with the activity of anionchannels than with other anion transport processes.