Ca2+-activated Cl- current in cultured myenteric neurons from murine proximal colon

Whole cell patch-clamprecordings were made from cultured myenteric neurons taken from murineproximal colon. The micropipette contained Cs⁺ to removeK⁺ currents. Depolarization elicited a slowly activatingtime-dependent outward current (I_tdo), whereasrepolarization was followed by a slowly deactivating tail current(I_tail). I_tdo andI_tail were present in ~70% of neurons. Weidentified these currents as Cl currents(I_Cl), because changing the transmembraneCl gradient altered the measured reversal potential(E_rev) of both I_tdo andI_tail with that for I_tailshifted close to the calculated Cl equilibrium potential(E_Cl). I_Cl areCa²⁺-activated Cl current[I_Cl(Ca)] because they were Ca²⁺dependent. E_Cl, which was measured from theE_rev of I_Cl(Ca) using agramicidin perforated patch, was 33 mV. This value is more positivethan the resting membrane potential (56.3 ± 2.7 mV), suggestingmyenteric neurons accumulate intracellular Cl.-Conotoxin GIVA [0.3 µM; N-type Ca²⁺ channelblocker] and niflumic acid [10 µM; knownI_Cl(Ca) blocker], decreased theI_Cl(Ca). In conclusion, these neurons haveI_Cl(Ca) that are activated by Ca²⁺entry through N-type Ca²⁺ channels. These currents likelyregulate postspike frequency adaptation.

     

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.

     

Ca2+ influx inhibits voltage-dependent and augments Ca2+-dependent K+ currents in arterial myocytes

These experiments were performed to determine the effects ofreducing Ca²⁺ influx(Ca_in) onK⁺ currents(I_K) inmyocytes from rat small mesenteric arteries by1) adding externalCd²⁺ or2) lowering externalCa²⁺ to 0.2 mM. When measured froma holding potential (HP) of 20 mV(I_K20),decreasing Ca_in decreasedI_K at voltageswhere it was active (>0 mV). When measured from a HP of 60 mV(I_K60),decreasing Ca_in increasedI_K at voltagesbetween 30 and +20 mV but decreased I_K at voltagesabove +40 mV. Difference currents(I_K) weredetermined by digital subtraction of currents recorded under controlconditions from those obtained whenCa_in was decreased. At testvoltages up to 0 mV,I_K60 exhibitedkinetics similar to controlI_K60, with rapidactivation to a peak followed by slow inactivation. At 0 mV, peakI_K60 averaged75 ± 13 pA (n = 8) withCd²⁺ and 120 ± 20 pA(n = 9) with lowCa²⁺ concentration. At testvoltages from 0 to +60 mV,I_K60 always had an early positive peak phase, but its apparent "inactivation" increased with voltage and its steady value became negative above +20mV. At +60 mV, the initial peakI_K60 averaged115 ± 18 pA with Cd²⁺ and 187 ± 34 pA with low Ca²⁺. With 10 mM pipette BAPTA, Cd²⁺ produced asmall inhibition ofI_K20 but stillincreased I_K60 between 30 and +10 mV. InCa²⁺-free external solution,Cd²⁺ only decreased bothI_K20 andI_K60. In thepresence of iberiotoxin (100 nM) to inhibitCa²⁺-activatedK⁺ channels(K_Ca),Cd²⁺ increasedI_K60 at allvoltages positive to 30 mV while BAY K 8644 (1 µM) decreasedI_K60. Theseresults suggest that Ca_in, through L-type Ca²⁺ channels and perhapsother pathways, increases K_Ca(i.e., I_K20) and decreases voltage-dependent K⁺currents in this tissue. This effect could contribute to membrane depolarization and force maintenance.

     

Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells

Patch-clamping and cell imageanalysis techniques were used to study the expression of thevolume-activated Cl current,I_Cl(vol), and regulatory volume decrease (RVD)capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated aCl current with a linear conductance, negligibletime-dependent inactivation, and a reversal potential close to theCl equilibrium potential. The sequence of anionpermeability was I > Br > Cl > gluconate. The Cl channelblockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),and ATP inhibited I_Cl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by adouble chemical-block (thymidine and hydroxyurea) technique. Theexpression of I_Cl(vol) was cell cycle dependent,being high in G₁ phase, downregulated in S phase, butincreasing again in M phase. Hypotonic solution activated RVD, whichwas cell cycle dependent and inhibited by the Cl channelblockers NPPB, tamoxifen, and ATP. The expression of I_Cl(vol) was closely correlated with the RVDcapacity in the cell cycle, suggesting a functional relationship.Inhibition of I_Cl(vol) by NPPB (100 µM)arrested cells in G₀/G₁. The data also suggest that expression of I_Cl(vol) and RVD capacity areactively modulated during the cell cycle. The volume-activatedCl current associated with RVD may therefore play animportant role during the cell cycle progress.

     

Role of gelsolin in the actin filament regulation of cardiac L-type calcium channels

The actin cytoskeleton is an important contributor to themodulation of the cell function. However, little is known about theregulatory role of this supermolecular structure in the membrane eventsthat take place in the heart. In this report, the regulation of cardiacmyocyte function by actin filament organization was investigated inneonatal mouse cardiac myocytes (NMCM) from both wild-type mice andmice genetically devoid of the actin filament severing protein gelsolin(Gsn/). Cardiac L-type calcium channel currents(I_Ca) wereassessed using the whole cell voltage-clamp technique. Addition of theactin filament stabilizer phalloidin to wild-type NMCM increasedI_Ca by 227% overcontrol conditions. The basalI_Ca ofGsn/ NMCM was 300% higher than wild-type controls. Thisincrease was completely reversed by intracellular perfusion of theGsn/ NMCM with exogenous gelsolin. Further, cytoskeletal disruption of either Gsn/ or phalloidin-dialyzedwild-type NMCM with cytochalasin D (CD) decreased the enhancedI_Ca by 84% and 87%, respectively. The data indicate that actin filament stabilization by either a lack of gelsolin or intracellular dialysis with phalloidin increase I_Ca,whereas actin filament disruption with CD or dialysis ofGsn/ NMCM with gelsolin decreaseI_Ca. We concludethat cardiac L-type calcium channel regulation is tightly controlled byactin filament organization. Actin filament rearrangement mediated by gelsolin may contribute to calcium channel inactivation.

     

Volume-regulated chloride conductance in the LNCaP human prostate cancer cell line

Patch-clamp recordings were used to study ioncurrents induced by cell swelling caused by hypotonicity in humanprostate cancer epithelial cells, LNCaP. The reversal potential of the swelling-evoked current suggested that Cl was the primarycharge carrier (termed I_Cl,swell). Theselectivity sequence of the underlying volume-regulated anion channels(VRACs) for different anions wasBrI > Cl > F > methanesulfonate glutamate, with relativepermeability numbers of 1.26, 1.20, 1.0, 0.77, 0.49, and 0.036, respectively. The current-voltage patterns of the whole cell currentsas well as single-channel currents showed moderate outwardrectification. Unitary VRAC conductance was determined at 9.6 ± 1.8 pS. Conventional Cl channel blockers5-nitro-2-(3-phenylpropylamino)benzoic acid (100 µM) and DIDS (100 µM) inhibited whole cell I_Cl,swell in a voltage-dependent manner, with the block decreasing from 39.6 ± 9.7% and 71.0 ± 11.0% at +50 mV to 26.2 ± 7.2% and14.5 ± 6.6% at 100 mV, respectively. Verapamil (50 µM), astandard Ca²⁺ antagonist and P-glycoprotein functioninhibitor, depressed the current by a maximum of 15%. Protein tyrosinekinase inhibitors downregulated I_Cl,swell(genistein with an IC₅₀ of 2.6 µM and lavendustin A by60 ± 14% at 1 µM). The protein tyrosine phosphatase inhibitorsodium orthovanadate (500 µM) stimulatedI_Cl,swell by 54 ± 11%. We conclude thatVRACs in human prostate cancer epithelial cells are modulated viaprotein tyrosine phosphorylation.

     

Deficiency in parvalbumin increases fatigue resistance in fast-twitch muscle and upregulates mitochondria

The solubleCa²⁺-binding protein parvalbumin (PV) is expressed at highlevels in fast-twitch muscles of mice. Deficiency of PV in knockoutmice (PV /) slows down the speed of twitch relaxation, whilemaximum force generated during tetanic contraction is unaltered. Weobserved that PV-deficient fast-twitch muscles were significantly moreresistant to fatigue than were the wild type. Thus components involvedin Ca²⁺ homeostasis during the contraction-relaxation cyclewere analyzed. No upregulation of another cytosolicCa²⁺-binding protein was found. Mitochondria are thought toplay a physiological role during muscle relaxation and were thusanalyzed. The fractional volume of mitochondria in the fast-twitchmuscle extensor digitorum longus (EDL) was almost doubled in PV /mice, and this was reflected in an increase of cytochrome coxidase. A faster removal of intracellular Ca²⁺concentration ([Ca²⁺]_i) 200-700 ms afterfast-twitch muscle stimulation observed in PV / muscles supportsthe role for mitochondria in late [Ca²⁺]_iremoval. The present results also show a significant increase of thedensity of capillaries in EDL muscles of PV / mice. Thus alterations in the dynamics of Ca²⁺ transients detected infast-twitch muscles of PV / mice might be linked to the increase inmitochondria volume and capillary density, which contribute to thegreater fatigue resistance of these muscles.

     

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.

     

Ca(2+)-activated Cl(-) current in sheep lymphatic smooth muscle

Freshly dispersed sheep mesenteric lymphaticsmooth muscle cells were studied at 37°C using the perforatedpatch-clamp technique with Cs⁺- and K⁺-filledpipettes. Depolarizing steps evoked currents that consisted ofL-type Ca²⁺ [I_Ca(L)]current and a slowly developing current. The slow current reversed at1 ± 1.5 mV with symmetrical Cl concentrationscompared with 23.2 ± 1.2 mV (n = 5) and34.3 ± 3.5 mV (n = 4) when externalCl was substituted with either glutamate (86 mM) orI (125 mM). Nifedipine (1 µM) blocked and BAY K 8644 enhanced I_Ca(L), the slow-developing sustainedcurrent, and the tail current. The Cl channel blockeranthracene-9-carboxylic acid (9-AC) reduced only the slowly developinginward and tail currents. Application of caffeine (10 mM) tovoltage-clamped cells evoked currents that reversed close to theCl equilibrium potential and were sensitive to 9-AC.Small spontaneous transient depolarizations and larger actionpotentials were observed in current clamp, and these were blocked by9-AC. Evoked action potentials were triphasic and had a prominentplateau phase that was selectively blocked by 9-AC. Similarly, fluidoutput was reduced by 9-AC in doubly cannulated segments ofspontaneously pumping sheep lymphatics, suggesting that theCa²⁺-activated Cl current plays an importantrole in the electrical activity underlying spontaneous activity in this tissue.

     

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.

     

Na+ pump alpha 2-subunit expression modulates Ca2+ signaling

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.

     

Potent inhibition of the aortic smooth muscle maxi-K channel by clinical doses of ethanol

We investigated the effects ofclinically relevant ethanol concentrations (5-20 mM) on thesingle-channel kinetics of bovine aortic smooth muscle maxi-K channelsreconstituted in lipid bilayers (1:1palmitoyl-oleoyl-phosphatidylethanolamine:palmitoyl-oleoyl-phosphatidylcholine). Ethanol at 10 and 20 mMdecreased the channel open probability (P_o) by75 ± 20.3% mainly by increasing the mean closed time (+82 to+960%, n = 7). In some instances, ethanol alsodecreased the mean open time (40.8 ± 22.5%). TheP_o-voltage relation in the presence of 20 mMethanol exhibited a rightward shift in the midpoint of voltageactivation (V_1/2  17 mV), a slightlysteeper relationship (change in slope factor, k,  2.5 mV), and a decreased maximum P_o (from~0.82 to ~0.47). Interestingly, channels inhibited by ethanol atlow Ca²⁺ concentrations (2.5 µM) were veryresistant to ethanol in the presence of increased Ca²⁺ ( 20 µM). Alcohol consumption in clinically relevant amounts may alterthe contribution of maxi-K channels to the regulation of arterial tone.

     

Evidence for functional role of epsilonPKC isozyme in the regulation of cardiac Na(+) channels

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 (I_Na) was recorded usingtwo-electrode voltage clamp. V1-7 (100 nM) and PMA (100 nM)inhibited I_Na 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 I_Na inhibition was abolished byV1-2, a peptide-specific antagonist of PKC. Furthermore,PMA-induced I_Na 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 I_Na; thisinhibition was antagonized by V1-2. In conclusion, the resultsprovide evidence for selective regulation of cardiac Na⁺channels by PKC isozyme.

     

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.

     

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.

     

Alterations in airway ion transport in NKCC1-deficient mice

Airways of Na⁺-K⁺-2Cl(NKCC1)-deficient mice (/) were studied in Ussing chambers todetermine the role of the basolateral NKCC1 in transepithelial anionsecretion. The basal short-circuit current (I_sc)of tracheae and bronchi from adult mice did not differ betweenNKCC1/ and normal mice, whereas NKCC1/ tracheae from neonatalmice exhibited a significantly reduced basalI_sc. In normal mouse tracheae, sensitivity tothe NKCC1 inhibitor bumetanide correlated inversely with the age of themouse. In contrast, tracheae from NKCC1/ mice at all ages wereinsensitive to bumetanide. The anion secretory response to forskolindid not differ between normal and NKCC1/ tissues. However, whenlarger anion secretory responses were induced with UTP, airways fromthe NKCC1/ mice exhibited an attenuated response. Ion substitutionand drug treatment protocols suggested that HCOsecretion compensated for reduced Cl secretion inNKCC1/ airway epithelia. The absence of spontaneous airway diseaseor pathology in airways from the NKCC1/ mice suggests that theNKCC1 mutant mice are able to compensate adequately for absence of theNKCC1 protein.

     

Adrenergic stimulation of Na+ transport across alveolar epithelial cells involves activation of apical Cl- channels

Alveolar epithelial cells were isolated from adultSprague-Dawley rats and grown to confluence on membrane filters. Mostof the basal short-circuit current(I_sc; 60%) wasinhibited by amiloride (IC₅₀ 0.96 µM) or benzamil (IC₅₀ 0.5 µM).Basolateral addition of terbutaline (2 µM) produced a rapid decreasein I_sc, followed by a slow recovery back to its initial amplitude. WhenCl was replaced withmethanesulfonic acid, the basalI_sc 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.

     

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.

     

Protein kinase C{varepsilon} contributes to regulation of the sarcolemmal Na+-K+ pump

A reduction in angiotensinII (ANG II) in vivo by treatment of rabbits with theangiotensin-converting enzyme inhibitor, captopril, increasesNa⁺-K⁺ pump current (I_p)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 I_p of myocytes voltageclamped with wide-tipped patch pipettes. I_p ofmyocytes from captopril-treated rabbits was larger thanI_p of myocytes from controls. ANG II superfusionof myocytes from captopril-treated rabbits decreasedI_p 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 I_p 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.

     

Ouabain and substrate affinities of human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells

HumanNa⁺-K⁺-ATPase₁₁,₂₁, and ₃₁heterodimers were expressed individually in yeast, and ouabainbinding and ATP hydrolysis were measured in membrane fractions. Theouabain equilibrium dissociation constant was 13-17 nM for₁₁ and ₃₁at 37°C and 32 nM for ₂₁, indicatingthat the human -subunit isoforms have a similar high affinity forcardiac glycosides. K_0.5 values for antagonism of ouabain binding by K⁺ were ranked in order as follows:₂ (6.3 ± 2.4 mM) > ₃(1.6 ± 0.5 mM)  ₁ (0.9 ± 0.6 mM),and K_0.5 values for Na⁺ antagonismof ouabain binding to all heterodimers were 9.5-13.8 mM. Themolecular turnover for ATP hydrolysis by₁₁ (6,652 min¹) was abouttwice as high as that by ₃₁ (3,145 min¹). These properties of the human heterodimersexpressed in yeast are in good agreement with properties of the humanNa⁺-K⁺-ATPase expressed in Xenopusoocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-DHorisberger, L Lelievie, and K Geering. J Biol Chem275: 1976-1986, 2000). In contrast to Na⁺ pumpsexpressed in Xenopus oocytes, the₂₁ complex in yeast membranes wassignificantly less stable than ₁₁ or₃₁, resulting in a lower functionalexpression level. The ₂₁ complex was also more easily denatured by SDS than was the₁₁ or the₃₁ complex.