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.

     

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.

     

Protein kinase Calpha participates in activation of store-operated Ca2+ channels in human glomerular mesangial cells

Protein kinase C (PKC) plays animportant role in activating store-operated Ca²⁺ channels(SOC) in human mesangial cells (MC). The present study was performed todetermine the specific isoform(s) of conventional PKC involved inactivating SOC in MC. Fura 2 fluorescence ratiometry showed that thethapsigargin-induced Ca²⁺ entry (equivalent to SOC) wassignificantly inhibited by 1 µM Gö-6976 (a specific PKC andI inhibitor) and PKC antisense treatment (2.5 nM for 24-48h). However, LY-379196 (PKC inhibitor) and2,2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethanoldimethyl ether(HBDDE; PKC and  inhibitor) failed to affect thapsigargin-evoked activation of SOC. Single-channel analysis in the cell-attached configuration revealed that Gö-6976 and PKC antisensesignificantly depressed thapsigargin-induced activation of SOC.However, LY-379196 and HBDDE did not affect the SOC responses. Ininside-out patches, application of purified PKC or I, but notII or , significantly rescued SOC from postexcision rundown.Western blot analysis revealed that thapsigargin evoked a decrease incytosolic expression with a corresponding increase in membraneexpression of PKC and . However, the translocation from cytosolto membranes was not detected for PKCI or II. These resultssuggest that PKC participates in the intracellular signaling pathwayfor activating SOC upon release of intracellular stores ofCa²⁺.

     

Upregulation of alpha(8)beta(1)-integrin in cardiac fibroblast by angiotensin II and transforming growth factor-beta1

Using a novel pharmacological tool with¹²⁵I-echistatin to detect integrins on the cell, we haveobserved that cardiac fibroblasts harbor five different RGD-bindingintegrins: ₈₁,₃₁, ₅₁, _v1, and _v3.Stimulation of cardiac fibroblasts by angiotensin II (ANG II) ortransforming growth factor-1 (TGF-1) resulted in an increase ofprotein and heightening by 50% of the receptor density of₈₁-integrin. The effect of ANG II wasblocked by an AT₁, but not an AT₂, receptorantagonist, or by an anti-TGF-1 antibody. ANG II and TGF-1increased fibronectin secretion, smooth muscle -actin synthesis, andformation of actin stress fibers and enhanced attachment of fibroblaststo a fibronectin matrix. The ₈- and₁-subunits were colocalized by immunocytochemistry with vinculin or ₃-integrin at focal adhesion sites.These results indicate that ₈₁-integrinis an abundant integrin on rat cardiac fibroblasts. Its positivemodulation by ANG II and TGF-1 in a myofibroblast-likephenotype suggests the involvement of₈₁-integrin in extracellularmatrix protein deposition and cardiac fibroblast adhesion.

     

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.

     

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.

     

Vascular endothelial cells express isoforms of protein kinase A inhibitor

First published September 5, 2001;10.1152/ ajpcell.00256.2001.The expression and function of theendogenous inhibitor of cAMP-dependent protein kinase (PKI) inendothelial cells are unknown. In this study, overexpression of rabbitmuscle PKI gene into endothelial cells inhibited the cAMP-mediatedincrease and exacerbated thrombin-induced decrease in endothelialbarrier function. We investigated PKI expression in human pulmonaryartery (HPAECs), foreskin microvessel (HMECs), and brain microvesselendothelial cells (HBMECs). RT-PCR using specific primers for humanPKI, human PKI, and mouse PKI sequences detectedPKI and PKI mRNA in all three cell types. Sequencing and BLASTanalysis indicated that forward and reverse DNA strands for PKI andPKI were of >96% identity with database sequences. RNaseprotection assays showed protection of the 542 nucleotides in HBMEC andHPAEC PKI mRNA and 240 nucleotides in HBMEC, HPAEC, and HMEC PKImRNA. Western blot analysis indicated that PKI protein was detectedin all three cell types, whereas PKI was found in HBMECs. Insummary, endothelial cells from three different vascular beds expressPKI and PKI, which may be physiologically important inendothelial barrier function.

     

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 overload increases beta-MHC promoter activity in rodent fast muscle via the proximal MCAT (betae3) site

Functional overload (OL)of the rat plantaris muscle by the removal of synergistic musclesinduces a shift in the myosin heavy chain (MHC) isoform expressionprofile from the fast isoforms toward the slow type I, or, -MHCisoform. Different length rat -MHC promoters were linked to afirefly luciferase reporter gene and injected in control and OLplantaris muscles. Reporter activities of 3,500, 914, 408, and215 bp promoters increased in response to 1 wk of OL. The smallest171 bp promoter was not responsive to OL. Mutation analyses ofputative regulatory elements within the 171 and 408 bp region wereperformed. The 408 bp promoters containing mutations of the e1,distal muscle CAT (MCAT; e2), CACC, or A/T-rich (GATA), were stillresponsive to OL. Only the proximal MCAT (e3) mutation abolished theOL response. Gel mobility shift assays revealed a significantly higherlevel of complex formation of the e3 probe with nuclear protein fromOL plantaris compared with control plantaris. These results suggestthat the e3 site functions as a putative OL-responsive element inthe rat -MHC gene promoter.

     

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.

     

Polyamines regulate beta-catenin tyrosine phosphorylation via Ca(2+) during intestinal epithelial cell migration

Polyaminesare essential for early mucosal restitution that occurs by epithelialcell migration to reseal superficial wounds after injury. Normalintestinal epithelial cells are tightly bound in sheets, but they needto be rapidly disassembled during restitution. -Catenin is involvedin cell-cell adhesion, and its tyrosine phosphorylation causesdisassembly of adhesion junctions, enhancing the spreading of cells.The current study determined whether polyamines are required for thestimulation of epithelial cell migration by altering -catenintyrosine phosphorylation. Migration of intestinal epithelial cells(IEC-6 line) after wounding was associated with an increase in-catenin tyrosine phosphorylation, which decreased the bindingactivity of -catenin to -catenin. Polyamine depletion by-difluoromethylornithine reduced cytoplasmic free Ca²⁺concentration ([Ca²⁺]_cyt), preventedinduction of -catenin phosphorylation, and decreased cell migration.Elevation of [Ca²⁺]_cyt induced by theCa²⁺ ionophore ionomycin restored -cateninphosphorylation and promoted migration in polyamine-deficient cells.Decreased -catenin phosphorylation through the tyrosine kinaseinhibitor herbimycin-A or genistein blocked cell migration, which wasaccompanied by reorganization of cytoskeletal proteins. These resultsindicate that -catenin tyrosine phosphorylation plays a criticalrole in polyamine-dependent cell migration and that polyamines induce-catenin tyrosine phosphorylation at least partially through[Ca²⁺]_cyt.

     

Direct estimate of 1:1 stoichiometry of K(+)-Cl(-) cotransport in rabbit erythrocytes

This work was undertaken toobtain a direct measure of the stoichiometry ofNa⁺-independent K⁺-Cl cotransport(KCC), with rabbit red blood cells as a model system. To determinewhether ⁸⁶Rb⁺ can be used quantitatively as atracer for KCC, ⁸⁶Rb⁺ and K⁺effluxes were measured in parallel after activation of KCC with N-ethylmaleimide (NEM). The rate constant for NEM-stimulatedK⁺ efflux into isosmotic NaCl was smaller than that for⁸⁶Rb⁺ by a factor of 0.68 ± 0.11 (SD,n = 5). This correction factor was used in all otherexperiments to calculate the K⁺ efflux from the measured⁸⁶Rb⁺ efflux. To minimize interference from theanion exchanger, extracellular Cl was replaced withSO, and4,4'-diisothiocyanothiocyanatodihydrostilbene-2,2'-disulfonic acid was present in the flux media. The membrane potential was clampednear 0 mV with the protonophore 2,4-dinitrophenol. The Clefflux at 25°C under these conditions is ~100,000-fold smaller thanthe uninhibited Cl/Cl exchange flux and isstimulated ~2-fold by NEM. The NEM-stimulated ³⁶Cl flux is inhibited by okadaic acid andcalyculin A, as expected for KCC. The ratio of the NEM-stimulatedK⁺ to Cl efflux is 1.12 ± 0.26 (SD,n = 5). We conclude thatK⁺-Cl cotransport in rabbit red blood cellshas a stoichiometry of 1:1.

     

Adult alveolar epithelial cells express multiple subtypes of voltage-gated K+ channels that are located in apical membrane

Whole cell perforated patch-clampexperiments were performed with adult rat alveolar epithelial cells.The holding potential was 60 mV, and depolarizing voltage stepsactivated voltage-gated K⁺ (Kv) channels. Thevoltage-activated currents exhibited a mean reversal potential of 32mV. Complete activation was achieved at 10 mV. The currents exhibitedslow inactivation, with significant variability in the time coursebetween cells. Tail current analysis revealed cell-to-cell variabilityin K⁺ selectivity, suggesting contributions of multiple Kv-subunits to the whole cell current. The Kv channels also displayedsteady-state inactivation when the membrane potential was held atdepolarized voltages with a window current between 30 and 5 mV.Analysis of RNA isolated from these cells by RT-PCR revealed thepresence of eight Kv -subunits (Kv1.1, Kv1.3, Kv1.4, Kv2.2, Kv4.1,Kv4.2, Kv4.3, and Kv9.3), three -subunits (Kv1.1, Kv2.1, andKv3.1), and two K⁺ channel interacting protein (KChIP)isoforms (KChIP2 and KChIP3). Western blot analysis with available Kv-subunit antibodies (Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3) showedlabeling of 50-kDa proteins from alveolar epithelial cells grown inmonolayer culture. Immunocytochemical analysis of cells from monolayersshowed that Kv1.1, Kv1.3, Kv1.4, Kv4.2, and Kv4.3 were localized to theapical membrane. We conclude that expression of multiple Kv -, -,and KChIP subunits explains the variability in inactivation gating andK⁺ selectivity observed between cells and that Kv channelsin the apical membrane may contribute to basal K⁺ secretionacross the alveolar epithelium.

     

Role of beta 1- and beta 3-adrenoceptors in the regulation of lipolysis and thermogenesis in rat brown adipocytes

To evaluate the physiological functions of₁-,₂-, and₃-adrenoceptors (ARs) in brownadipose tissue, the lipolytic and respiratory effects of variousadrenergic agonists and antagonists were studied in rat brownadipocytes. The -agonists stimulated both lipolysis and respiration(8-10 times above basal levels), with the following order ofpotency (concentration eliciting 50% of maximum response):CL-316243 (₃) > BRL-37344(₃) > isoproterenol (mainly₁/₂) > norepinephrine (NE; mainly₁/₂) > epinephrine (mainly₁/₂) dobutamine (₁)  procaterol (₂). Schild plot coefficients of competitive inhibition experiments using ICI-89406 (₁ antagonist) revealed thatmore than one type of receptor mediates NE action. It is concluded fromour results that 1) NE, at low plasma levels (1-25 nM), stimulates lipolysis and respiration mainly through ₁-ARs,2) NE, at higher levels, stimulateslipolysis and respiration via both₁- and₃-ARs,3)₂-ARs play only a minor role,and 4)₃-ARs may represent thephysiological receptors for the high NE concentrations in the synapticcleft, where the high-affinity₁-ARs are presumablydesensitized. It is also suggested that lipolysis represents theflux-generating step regulating mitochondrial respiration.

     

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.

     

Three-dimensional tissue structure affects sensitivity of fibroblasts to TGF-beta 1

Transforming growth factor-(TGF-) is known to induce -smooth muscle actin (-SMA) infibroblasts and is supposed to play a role in myofibroblastdifferentiation and tumor desmoplasia. Our objective was to elucidatethe impact of TGF-1 on -SMA expression in fibroblasts in athree-dimensional (3-D) vs. two-dimensional (2-D) environment. Inmonolayer culture, all fibroblast cultures responded in a similarfashion to TGF-1 with regard to -SMA expression. In fibroblastspheroids, -SMA expression was reduced and induction by TGF-1 washighly variable. This difference correlated with a differentialregulation in the TGF- receptor (TGFR) expression, in particularwith a reduction in TGF-RII in part of the fibroblast types. Ourdata indicate that 1) sensitivity to TGF-1-induced -SMA expression in a 3-D environment is fibroblast-type specific, 2) fibroblast type-independent regulatory mechanisms, suchas a general reduction/loss in TGF-RIII, contribute to an altered TGFR expression profile in spheroid compared with monolayer culture, and 3) fibroblast type-specific alterations in TGFR typesI and II determine the sensitivity to TGF-1-induced -SMAexpression in the 3-D setting. We suggest that fibroblasts that can beinduced by TGF-1 to produce -SMA in spheroid culture reflect a"premyofibroblastic" phenotype.

     

Essential role of ICAM-1 in mediating monocyte adhesion to aortic endothelial cells

Monocyte-endothelial cell interactions havebeen implicated in the pathogenesis of a number of vascular diseasesthat target arterial and aortic endothelium, including atherosclerosis.Many different adhesion molecules, such as intercellular adhesionmolecule (ICAM)-1, are thought to mediate monocyte binding toendothelial cells during the development of these diseases. However,conflicting results have been reported regarding the specific role ofICAM-1 in these events. In this study, we used a genetic approach to determine the contribution of ICAM-1 in mediating monocyte adhesion tomouse aortic endothelial cells (MAEC) derived from both wild-type andICAM-1^/ mice. Treatment of wild-type MAEC with oxidizedlow-density lipoprotein significantly induced both WEHI 274.1 and wholeblood monocyte adhesion, whereas similarly treatedICAM-1^/ MAEC showed a complete inhibition of monocytebinding. Dose-response treatment with tumor necrosis factor- alsoincreased monocyte adhesion to wild-type MAEC, but significant adhesionwas only observed at higher doses for ICAM-1^/ MAEC.These data demonstrate a crucial role for ICAM-1-mediated monocyte-endothelial cell interactions in response to specific stimuliinvolved in inflammatory vascular diseases.

     

The gamma-subunit of ENaC is more important for channel surface expression than the beta-subunit

The amiloride-sensitiveepithelial sodium channel (ENaC) plays a critical role in fluid andelectrolyte homeostasis and is composed of three homologous subunits:, , and . Only heteromultimeric channels made of ENaCare efficiently expressed at the cell surface, resulting in maximallyamiloride-sensitive currents. To study the relative importance ofvarious regions of the - and -subunits for the expression offunctional ENaC channels at the cell surface, we constructedhemagglutinin (HA)-tagged --chimeric subunits composed of -and -subunit regions and coexpressed them with HA-tagged - and-subunits in Xenopus laevis oocytes. The whole cellamiloride-sensitive sodium current (I_ami) andsurface expression of channels were assessed in parallel using thetwo-electrode voltage-clamp technique and a chemiluminescence assay.Because coexpression of ENaC resulted in largerI_ami and surface expression compared withcoexpression of ENaC, we hypothesized that the -subunit ismore important for ENaC trafficking than the -subunit. Usingchimeras, we demonstrated that channel activity is largely preservedwhen the highly conserved second cysteine rich domains (CRD2) of the- and -subunits are exchanged. In contrast, exchanging the wholeextracellular loops of the - and the -subunits largely reducedENaC currents and ENaC expression in the membrane. This indicates thatthere is limited interchangeability between molecular regions of thetwo subunits. Interestingly, our chimera studies demonstrated that theintracellular termini and the two transmembrane domains of ENaC aremore important for the expression of functional channels at the cellsurface than the corresponding regions of ENaC.

     

Role of alpha(v)beta(3)-integrin in TNF-alpha-induced endothelial cell migration

Tumor necrosis factor- (TNF-), oneof the major inflammatory cytokines, is known to influence endothelialcell migration. In this study, we demonstrate that exposure of calfpulmonary artery endothelial cells to TNF- caused an increase in theformation of membrane protrusions and cell migration. Fluorescencemicroscopy revealed an increase in _v3focal contacts but a decrease in ₅₁ focalcontacts in TNF--treated cells. In addition, both cell-surface andtotal cellular expression of _v3-integrinsincreased significantly, whereas the expression of₅₁-integrins was unaltered. Only focalcontacts containing _v3- but not₅₁-integrins were present in membraneprotrusions of cells at the migration front. In contrast, robust focalcontacts containing ₅₁-integrins were present in cells behind the migration front. A blocking antibody to_v3, but not a blocking antibody to₅-integrins, significantly inhibited TNF--inducedcell migration. These results indicate that in response to TNF-,endothelial cells may increase the activation and ligation of_v3 while decreasing the activation andligation of ₅₁-integrins to facilitatecell migration, a process essential for vascular wound healing and angiogenesis.

     

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

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