Shear stress induces a time- and position-dependent increase in endothelial cell membrane fluidity

Blood flow-associatedshear stress may modulate cellular processes through its action on theplasma membrane. We quantified the spatial and temporal aspects of theeffects of shear stress () on the lipid fluidity of1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC₁₆(13)]-stained plasma membranesof bovine aortic endothelial cells in a flow chamber. A confocalmicroscope was used to determine the DiI diffusion coefficient(D) by fluorescence recovery after photobleaching on cellsunder static conditions, after a step- of 10 or 20 dyn/cm², and after the cessation of . The methodallowed the measurements of D on the upstream and downstreamsides of the cell taken midway between the respective cell borders andthe nucleus. In <10 s after a step- of 10 dyn/cm²,D showed an upstream increase and a downstream decrease, and both changes disappeared rapidly. There was a secondary, larger increase in upstream D, which reached a peak at 7 min and decreased thereafter, despite the maintenance of .D returned to near control values within 5 s aftercessation of . Downstream D showed little secondarychanges throughout the 10-min shearing, as well as after its cessation.Further investigations into the early phase, with simultaneousmeasurements of upstream and downstream D, confirmed that astep- of 10 dyn/cm² elicited a rapid (5-s) but transientincrease in upstream D and a concurrent decrease indownstream D, yielding a significant difference between thetwo sites. A step- of 20 dyn/cm² caused D toincrease at both sites at 5 s, but by 30 s and 1 min theupstream D became significantly higher than the downstream D. These results demonstrate shear-induced changes inmembrane fluidity that are time dependent and spatially heterogeneous. These changes in membrane fluidity may have important implications inshear-induced membrane protein modulation.

     

IkappaBalpha-dependent regulation of low-shear flow-induced NF-kappa B activity: role of nitric oxide

We have investigated the role ofinhibitor B (IB) in the activation of nuclear factor B(NF-B) observed in human aortic endothelial cells (HAEC) undergoinga low shear stress of 2 dynes/cm². Low shear for 6 hresulted in a reduction of IB levels, an activation of NF-B,and an increase in B-dependent vascular cell adhesion molecule 1 (VCAM-1) mRNA expression and endothelial-monocyte adhesion.Overexpression of IB in HAEC attenuated all of these shear-induced responses. These results suggest that downregulation ofIB is the major factor in the low shear-induced activation ofNF-B in HAEC. We then investigated the role of nitric oxide (NO) inthe regulation of IB/NF-B. Overexpression of endothelial nitric oxide synthase (eNOS) inhibited NF-B activation in HAEC exposed to 6 h of low shear stress. Addition of the structurally unrelated NO donors S-nitrosoglutathione (300 µM) orsodium nitroprusside (1 mM) before low shear stress significantlyincreased cytoplasmic IB and concomitantly reduced NF-Bbinding activity and B-dependent VCAM-1 promoter activity. Together,these data suggest that NO may play a major role in the regulation ofIB levels in HAEC and that the application of low shear flowincreases NF-B activity by attenuating NO generation and thusIB levels.

     

Flow-induced expression of endothelial Na-K-Cl cotransport: dependence on K(+) and Cl(-) channels

Steady laminarshear stress has been shown previously to markedly increase Na-K-Clcotransporter mRNA and protein in human umbilical vein endothelialcells and also to rapidly increase endothelial K⁺ andCl channel conductances. The present study was done toevaluate the effects of shear stress on Na-K-Cl cotransporter activity and protein expression in bovine aortic endothelial cells (BAEC) and todetermine whether changes in cotransporter expression may be dependenton early changes in K⁺ and Cl channelconductances. Confluent BAEC monolayers were exposed in aparallel-plate flow chamber to either steady shear stress (19 dyn/cm²) or purely oscillatory shear stress (0 ± 19 dyn/cm²) for 6-48 h. After shearing, BAEC monolayerswere assessed for Na-K-Cl cotransporter activity or were subjected toWestern blot analysis of cotransporter protein. Steady shear stress ledto a 2- to 4-fold increase in BAEC cotransporter protein levels and a1.5- to 1.8-fold increase in cotransporter activity, increases thatwere sustained over the longest time periods studied. Oscillatory flow,in contrast, had no effect on cotransporter protein levels. In thepresence of flow-sensitive K⁺ and Cl channelpharmacological blockers, the steady shear stress-induced increase incotransporter protein was virtually abolished. These results suggestthat shear stress modulates the expression of the BAEC Na-K-Clcotransporter by mechanisms that are dependent on flow-activated ion channels.

     

Colon carcinoma cell glycolipids,integrins, and other glycoproteins mediate adhesion to HUVECs under flow

This study was undertaken toinvestigate the molecular constituents mediating LS174T colonadenocarcinoma cell adhesion to 4-h TNF--stimulated human umbilicalvein endothelial cells (HUVECs) under flow. At 1 dyn/cm²,~57% of cells rolled and then became firmly adherent, whereas otherscontinuously rolled on endothelium. Initial cell binding was primarilymediated by endothelial E-selectin. By using neuraminidase, glycolipidbiosynthesis inhibitord,l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol · HCl,trypsin, and flow cytometry, LS174T cells were shown to express sialylLewis^x (sLe^x)- anddi-sLe^x-decorated, but not sLe^a-decorated,glycolipid and glycoprotein ligands for E-selectin. The cellspreferentially employed sialylated glycoproteins over glycolipids inadhesion as measured by conversion of rolling to firm adhesion,resistance to detachment by increased shear stress, and rollingvelocity. However, a nonsialylated E-selectin counterreceptor alsoexists. Furthermore, LS174T ₂, ₆, and₁ integrins support a minor pathway in adhesion toHUVECs. Finally, tumor cell attachment specifically increases HUVECendocytosis of E-selectin. Altogether, the data indicate the complexityof carcinoma cell-endothelium adhesion via sialylated glycoconjugates,integrins, and their respective counterreceptors.

     

Lung epithelial barrier function and wound healing are decreased by IL-4 and IL-13 and enhanced by IFN-gamma

To understand theeffects of cytokines on epithelial cells in asthma, we haveinvestigated the effects of interleukin (IL)-4, IL-13, and interferon(IFN)- on barrier function and wound healing in Calu-3 human lungepithelial cells. IL-4 and IL-13 treatment of Calu-3 cells grown onTranswell filters resulted in a 70-75% decrease in barrierfunction as assessed by electrophysiological and[¹⁴C]mannitol flux measurements. In contrast, IFN-enhanced barrier function threefold using these same parameters. Cellstreated concurrently with IFN- and IL-4 or IL-13 showed an initialdecline in barrier function that was reversed within 2 days, resulting in barrier levels comparable to control cells. Analysis of the tightjunction-associated proteins ZO-1 and occludin showed that IL-4 andIL-13 significantly reduced ZO-1 expression and modestly decreasedoccludin expression compared with controls. IFN-, quite unexpectedlygiven its enhancing effect on barrier function, reduced expression ofZO-1 and occludin to almost undetectable levels compared with controls.In wound-healing assays of cells grown on collagen I, IL-4 and IL-13decreased migration, whereas IFN- treatment enhanced migration,compared with control cells. Addition of IFN-, in combination withIL-4 or IL-13, restored migration of cells to control levels. Migrationdifferences observed between the various cytokine treatments wascorrelated with expression of the collagen I-binding₂₁-integrin at the leading edge of cellsat the wound front; ₂₁-integrinexpression was decreased in IFN--treated cells compared withcontrols, whereas it was highest in IL-4- and IL-13-treated cells.These results demonstrate that IL-4 and IL-13 diminish the capacity ofCalu-3 cells to maintain barrier function and repair wounds, whereasIFN- promotes epithelial restitution by enhancing barrier functionand wound healing.

     

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.

     

Effect of glutamine supplementation on exercise-induced changes in lymphocyte function

The purpose of this study was toinvestigate the possible role of glutamine in exercise-inducedimpairment of lymphocyte function. Ten male athletesparticipated in a randomized, placebo-controlled, double-blindcrossover study. Each athlete performed bicycle exercise for 2 hat 75% of maximum O₂ consumption on 2 separate days.Glutamine or placebo supplements were given orally during and up to2 h postexercise. The trial induced postexercise neutrocytosisthat lasted at least 2 h. The total lymphocyte count increased bythe end of exercise due to increase of bothCD3⁺TCR⁺ andCD3⁺TCR⁺ T cells as well asCD3CD16⁺CD56⁺ naturalkiller (NK) cells. Concentrations of CD8⁺ andCD4⁺ T cells lacking CD28 and CD95 on their surfaceincreased more than those of cells expressing these receptors. Withinthe CD4⁺ cells, only CD45RA memory cells, butnot CD45RA⁺ naive cells, increased in response to exercise.Most lymphocyte subpopulations decreased 2 h after exercise.Glutamine supplementation abolished the postexercise decline in plasmaglutamine concentration but had no effect on lymphocyte trafficking, NKand lymphokine-activated killer cell activities, T cell proliferation,catecholamines, growth hormone, insulin, or glucose. Neutrocytosis wasless pronounced in the glutamine-supplemented group, but it is unlikelythat this finding is of any clinical significance. This study does notsupport the idea that glutamine plays a mechanistic role inexercise-induced immune changes.

     

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.

     

Inhibitors of glycosphingolipid biosynthesis reduce transepithelial electrical resistance in MDCK I and FRT cells

Madin-Darby canine kidney (MDCK)I and Fisher rat thyroid (FRT) cells exhibit transepithelial electricalresistance (TER) values in excess of 5,000  · cm². When these cells wereincubated in the presence of various inhibitors of sphingolipidbiosynthesis, a >5-fold reduction of TER was observed without changesin the gate function for uncharged solutes or the fence function forapically applied fluorescent lipids. The localization of ZO-1 andoccludin was not altered between control and inhibitor-treated cells,indicating that the tight junction was still intact. Furthermore, thecomplexity of tight junction strands, analyzed by freeze-fracturemicroscopy, was not reduced. Once the inhibitor was removed and thecells were allowed to synthesize sphingolipids, a gradual recovery ofthe TER was observed. Interestingly, these inhibitors did not attenuatethe TER of MDCK II cells, a cell line that typically exhibits valuesbelow 800  · cm^2. These resultssuggest that glycosphingolipids play a role in regulating theelectrical properties of epithelial cells.

     

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.

     

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⁺.

     

Agonist-specific cross talk between ERKs and p38(mapk) regulates PGI(2) synthesis in endothelium

We have examined the mechanisms regulatingprostacyclin (PGI₂) synthesis after acute exposure of humanumbilical vein endothelial cells (HUVEC) to interleukin-1 (IL-1).IL-1 evoked an early (30 min) release of PGI₂ and[³H]arachidonate that was blocked by the cytosolicphospholipase A₂ (cPLA₂) inhibitorarachidonyl trifluoromethyl ketone. IL-1-mediated activationof extracellular signal-regulated kinase 1/2 (ERK1/2; p42/p44^mapk) coincided temporally with phosphorylation ofcPLA₂ and with the onset of PGI₂synthesis. The mitogen-activated protein kinase (MAPK) kinase (MEK)inhibitors, PD-98059 and U-0126, blocked IL-1-induced ERKactivation and partially attenuated cPLA₂phosphorylation and PGI₂ release, suggesting thatERK-dependent and -independent pathways regulate cPLA₂phosphorylation. SB-203580 treatment enhanced IL-1-induced MEK,p42/44^mapk, and cPLA₂ phosphorylation butreduced thrombin-stimulated MEK and p42/44^mapk activation.IL-1, but not thrombin, activated Raf-1 as assessed byimmune-complex kinase assay, as did SB-203580 alone. These results showthat IL-1 causes an acute upregulation of PGI₂generation in HUVEC, establish a role for theMEK/ERK/cPLA₂ pathway in this early release, and provideevidence for an agonist-specific cross talk between p38^mapkand p42/44^mapk that may reflect receptor-specificdifferences in the signaling elements proximal to MAPK activation.

     

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.

     

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.

     

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.

     

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.

     

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.

     

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.

     

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.

     

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.