Relationship between intracellular pH and chloride in Xenopus oocytes expressing the chloride channel ClC-0

During maturation of oocytes,Cl conductance (G_Cl) oscillatesand intracellular pH (pH_i) increases. ElevatingpH_i permits the protein synthesis essential to maturation.To examine whether changes in G_Cl andpH_i are coupled, the Cl channel ClC-0 washeterologously expressed. Overexpressing ClC-0 elevatespH_i, decreases intracellular Cl concentration([Cl]_i), and reduces volume. Acuteacidification with butyrate does not activate acid extrusion inClC-0-expressing or control oocytes. The ClC-0-induced pH_ichange increases after overnight incubation at extracellular pH 8.5 butis unaltered after incubation at extracellular pH 6.5. Membranedepolarization did not change pH_i. In contrast, hyperpolarization elevates pH_i. Thus neither membranedepolarization nor acute activation of acid extrusion accounts for theClC-0-dependent alkalinization. Overnight incubation in lowextracellular Cl concentration increases pH_iand decreases [Cl]_i in control and ClC-0expressing oocytes, with the effect greater in the latter. Incubationin hypotonic, low extracellular Cl solutions preventedpH_i elevation, although the decrease in[Cl]_i persisted. Taken together, ourobservations suggest that KCl loss leads to oocyte shrinkage, whichtransiently activates acid extrusion. In conclusion, expressing ClC-0in oocytes increases pH_i and decreases[Cl]_i. These parameters are coupled viashrinkage activation of proton extrusion. Normal, cyclical changes ofoocyte G_Cl may exert an effect onpH_i via shrinkage, thus inducing meiotic maturation.

     

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.

     

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.

     

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.

     

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.

     

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.

     

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.

     

PGE(2), Ca(2+), and cAMP mediate ATP activation of Cl(-) channels in pigmented ciliary epithelial cells

Purines regulate intraocular pressure. Adenosine activatesCl channels of nonpigmented ciliary epithelial cellsfacing the aqueous humor, enhancing secretion. Tamoxifen and ATPsynergistically activate Cl channels of pigmented ciliaryepithelial (PE) cells facing the stroma, potentially reducing netsecretion. The actions of nucleotides alone on Cl channelactivity of bovine PE cells were studied by electronic cell sorting,patch clamping, and luciferin/luciferase ATP assay. Clchannels were activated by ATP > UTP, ADP, and UDP, but not by 2-methylthio-ATP, all at 100 µM. UTP triggered ATP release. The second messengers Ca²⁺, prostaglandin (PG)E₂,and cAMP activated Cl channels without enhancing effectsof 100 µM ATP. Buffering intracellular Ca²⁺activity with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acidor blocking PGE₂ formation with indomethacininhibited ATP-triggered channel activation. The Rp stereoisomerof 8-bromoadenosine 3',5'-cyclic monophosphothioate inhibited proteinkinase A activity but mimicked 8-bromoadenosine 3',5'-cyclicmonophosphate. We conclude that nucleotides can act at >1 P2Yreceptor to trigger a sequential cascade involving Ca²⁺,PGE₂, and cAMP. cAMP acts directly on Clchannels of PE cells, increasing stromal release and potentially reducing net aqueous humor formation and intraocular pressure.

     

Contribution of chloride channels to volume regulation of cortical astrocytes

The objective of this study was todetermine the relative contribution of Cl channels tovolume regulation of cultured rat cortical astrocytes after hypotoniccell swelling. Using a Coulter counter, we showed that corticalastrocytes regulate their cell volume by ~60% within 45 min afterhypotonic challenge. This volume regulation was supported whenCl was replaced with Br,NO, methanesulfonate, oracetate but was inhibited when Cl wasreplaced with isethionate or gluconate.Additionally, substitution of Cl with Icompletely blocked volume regulation. Volume regulation was unaffected by furosemide or bumetanide, blockers of KCl transport, but was inhibited by Cl channel blockers, including5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and niflumicacid. Surprisingly, the combination of Cd²⁺ with NPPB,DIDS, or niflumic acid inhibited regulation to a greater extent thanany of these drugs alone. Volume regulation did not differ amongastrocytes cultured from different brain regions, as cerebellar andhippocampal astrocytes exhibited behavior identical to that of corticalastrocytes. These data suggest that Cl flux through ionchannels rather than transporters is essential for volume regulation ofcultured astrocytes in response to hypotonic challenge.

     

Role of JNK in hypertonic activation of Cl--dependent Na+/H+ exchange in Xenopus oocytes

In the course of studying the hypertonicity-activated iontransporters in Xenopus oocytes, we found that activation ofendogenous oocyte Na⁺/H⁺ exchange activity(xoNHE) by hypertonic shrinkage required Cl, with anEC₅₀ for bath [Cl] of ~3 mM. Thisrequirement for chloride was not supported by several nonhalide anionsand was not shared by xoNHE activated by acid loading.Hypertonicity-activated xoNHE exhibited an unusual rank order ofinhibitory potency among amiloride derivatives and was blocked byCl transport inhibitors. Chelation of intracellularCa²⁺ by injection of EGTA blocked hypertonic activation ofxoNHE, although many inhibitors of Ca²⁺-related signalingpathways were without inhibitory effect. Hypertonicity activated oocyteextracellular signal-regulated kinase 1/2 (ERK1/2), but inhibitors ofneither ERK1/2 nor p38 prevented hypertonic activation of xoNHE.However, hypertonicity also stimulated a Cl-dependentincrease in c-Jun NH₂-terminal kinase (JNK) activity. Inhibition of JNK activity prevented hypertonic activation of xoNHE butnot activation by acid loading. We conclude that hypertonic activationof Na⁺/H⁺ exchange in Xenopusoocytes requires Cl and is mediated by activation of JNK.

     

Bumetanide blocks CFTR GCl in the native sweat duct

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

     

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.

     

Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms

The phorbol ester phorbol12-myristate 13-acetate (PMA) inhibits Cl secretion(short-circuit current, I_sc) and decreasesbarrier function (transepithelial resistance, TER) in T84 epithelia. To elucidate the role of specific protein kinase C (PKC) isoenzymes inthis response, we compared PMA with two non-phorbol activators of PKC(bryostatin-1 and carbachol) and utilized three PKC inhibitors (Gö-6850, Gö-6976, and rottlerin) with different isozymeselectivity profiles. PMA sequentially inhibited cAMP-stimulatedI_sc and decreased TER, as measured byvoltage-current clamp. By subcellular fractionation and Western blot,PMA (100 nM) induced sequential membrane translocation of the novelPKC followed by the conventional PKC and activated both isozymesby in vitro kinase assay. PKC was activated by PMA but did nottranslocate. By immunofluorescence, PKC redistributed to thebasolateral domain in response to PMA, whereas PKC moved apically.Inhibition of I_sc by PMA was prevented by theconventional and novel PKC inhibitor Gö-6850 (5 µM) but not theconventional isoform inhibitor Gö-6976 (5 µM) or the PKCinhibitor rottlerin (10 µM), implicating PKC in inhibition ofCl secretion. In contrast, both Gö-6976 andGö-6850 prevented the decline of TER, suggesting involvement ofPKC. Bryostatin-1 (100 nM) translocated PKC and PKC andinhibited cAMP-elicited I_sc. However, unlikePMA, bryostatin-1 downregulated PKC protein, and the decrease in TERwas only transient. Carbachol (100 µM) translocated only PKC andinhibited I_sc with no effect on TER. Gö-6850 but not Gö-6976 or rottlerin blocked bryostatin-1and carbachol inhibition of I_sc. We concludethat basolateral translocation of PKC inhibits Clsecretion, while apical translocation of PKC decreases TER. Thesedata suggest that epithelial transport and barrier function can bemodulated by distinct PKC isoforms.

     

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.

     

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.

     

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.

     

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.

     

TGF-beta effects on epithelial ion transport and barrier: reduced Cl- secretion blocked by a p38 MAPK inhibitor

Growthfactors affect a variety of epithelial functions. We examined theability of TGF- to modulate epithelial ion transport andpermeability. Filter-grown monolayers of human colonic epithelia, T84and HT-29 cells, were treated with TGF- (0.1-100 ng/ml,15 min-72 h) or infected with an adenoviral vector encodingTGF- (Ad-TGF) for 144 h. Ion transport (i.e., short-circuitcurrent, I_sc) and transepithelial resistance(TER) were assessed in Ussing chambers. Neither recombinant TGF- norAd-TGF infection affected baseline I_sc;however, exposure to 1 ng/ml TGF- led to a significant (30-50%) reduction in the I_sc responses toforskolin, vasoactive intestinal peptide, and cholera toxin (agentsthat evoke Cl secretion via cAMP mobilization) and to thecell-permeant dibutyryl cAMP. Pharmacological analysis of signalingpathways revealed that the inhibition of cAMP-driven epithelialCl secretion by TGF- was blocked by pretreatment withSB-203580, a specific inhibitor of p38 MAPK, but not by inhibitors ofJNK, ERK1/2 MAPK, or phosphatidylinositol 3'-kinase. TGF- enhanced the barrier function of the treated monolayers by up to threefold asassessed by TER; however, this event was temporally displaced from thealtered I_sc response, being statisticallysignificant only at 72 h posttreatment. Thus, in addition toTGF- promotion of epithelial barrier function, we show that thisgrowth factor also reduces responsiveness to cAMP-dependentsecretagogues in a chronic manner and speculate that this serves as abraking mechanism to limit secretory enteropathies.

     

Regulation of ATP-sensitive K(+) channels by protein kinase C in murine colonic myocytes

We investigated the regulation ofATP-sensitive K⁺ (K_ATP) currents in murinecolonic myocytes with patch-clamp techniques. Pinacidil(10⁵ M) activated inward currents in the presence of highexternal K⁺ (90 mM) at a holding potential of 80 mV indialyzed cells. Glibenclamide (10⁵ M) suppressedpinacidil-activated current. Phorbol 12,13-dibutyrate (PDBu; 2 × 10⁷ M) inhibited pinacidil-activated current.4--Phorbol ester (5 × 10⁷ M), an inactive formof PDBu, had no effect on pinacidil-activated current. In cell-attachedpatches, the open probability of K_ATP channels wasincreased by pinacidil, and PDBu suppressed openings ofK_ATP channels. When cells were pretreated withchelerythrine (10⁶ M) or calphostin C (10⁷M), inhibition of the pinacidil-activated whole cell currents by PDBuwas significantly reduced. In cells studied with the perforated patchtechnique, PDBu also inhibited pinacidil-activated current, and thisinhibition was reduced by chelerythrine (10⁶ M).Acetylcholine (ACh; 10⁵ M) inhibited pinacidil-activatedcurrents, and preincubation of cells with calphostin C(10⁷ M) decreased the effect of ACh. Cells dialyzed withprotein kinase C -isoform (PKC) antibody had normal responses topinacidil, but the effects of PDBu and ACh on K_ATP wereblocked in these cells. Immunofluorescence and Western blots showedexpression of PKC in intact muscles and isolated smooth muscle cellsof the murine proximal colon. These data suggest that PKC regulates K_ATP in colonic muscle cells and that the effects of ACh onK_ATP are largely mediated by PKC. PKC appears to be themajor isozyme that regulates K_ATP in murine colonic myocytes.

     

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.