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.

     

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.

     

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⁺.

     

Arachidonic acid mediates dual effect of TNF-alpha on Ca2+ transients and contraction of adult rat cardiomyocytes

Tumor necrosisfactor (TNF)- has a biphasic effect on heart contractility andstimulates phospholipase A₂ (PLA₂) incardiomyocytes. Because arachidonic acid (AA) exerts a dual effect onintracellular Ca²⁺ concentration([Ca²⁺]_i) transients, we investigated thepossible role of AA as a mediator of TNF- on[Ca²⁺]_i transients and contraction withelectrically stimulated adult rat cardiac myocytes. At a lowconcentration (10 ng/ml) TNF- produced a 40% increase in theamplitude of both [Ca²⁺]_i transients andcontraction within 40 min. At a high concentration (50 ng/ml) TNF-evoked a biphasic effect comprising an initial positive effect peakingat 5 min, followed by a sustained negative effect leading to50-40% decreases in [Ca²⁺]_i transientsand contraction after 30 min. Both the positive and negative effects ofTNF- were reproduced by AA and blocked by arachidonyltrifluoromethylketone (AACOCF3), an inhibitor of cytosolic PLA₂.Lipoxygenase and cyclooxygenase inhibitors reproduced the high-doseeffects of TNF- and AA. The negative effects of TNF- and AA werealso reproduced by sphingosine and were abrogated by the ceramidaseinhibitor n-oleoylethanolamine. These results point out thekey role of the cytosolic PLA₂/AA pathway in mediating thecontractile effects of TNF-.

     

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.

     

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.

     

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.

     

Permeant but not impermeant divalent cations enhance activation of nondesensitizing alpha(7) nicotinic receptors

Neuronal₇ nicotinic acetylcholine receptors (nAChRs) arepermeable to Ca²⁺ and other divalent cations. Wecharacterized the modulation of the pharmacological properties ofnondesensitizing mutant (L²⁴⁷T andS²⁴⁰T/L²⁴⁷T) ₇ nAChRs bypermeant (Ca²⁺, Ba²⁺, and Sr²⁺) andimpermeant (Cd²⁺ and Zn²⁺) divalent cations.₇ receptors were expressed in Xenopus oocytes and studied with two-electrode voltage clamp. Extracellular permeant divalent cations increased the potency and maximal efficacy of ACh,whereas impermeant divalent cations decreased potency and maximalefficacy. The antagonist dihydro--erythroidine (DHE) was a strongpartial agonist of L²⁴⁷T andS²⁴⁰T/L²⁴⁷T ₇ receptors in thepresence of divalent cations but was a weak partial agonist in thepresence of impermeant divalent cations. Mutation of the"intermediate ring" glutamates (E²³⁷A) inL²⁴⁷T ₇ nAChRs eliminated Ca²⁺conductance but did not alter the Ca²⁺-dependent increasein ACh potency, suggesting that site(s) required for modulation are onthe extracellular side of the intermediate ring. The difference betweenpermeant and impermeant divalent cations suggests that sites within thepore are important for modulation by divalent cations.

     

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.

     

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.

     

Role of PKCalpha in feedback regulation of Na(+) transport in an electrically tight epithelium

It has long been known thatNa⁺ channels in electrically tight epithelia are regulatedby homeostatic mechanisms that maintain a steady state and allow newlevels of transport to be sustained in hormonally challenged cells.Little is known about the potential pathways involved in theseprocesses. In addition to short-term effect, recent evidence alsoindicates the involvement of PKC in the long-term regulation of theepithelial Na⁺ channel (ENaC) at the protein level(40). To determine whether stimulation of ENaC involvesfeedback regulation of PKC levels, we utilized Western blot analysis todetermine the distribution of PKC isoforms in polarized A6 epithelia.We found the presence of PKC isoforms in the conventional ( and), novel (, , and ), and atypical (, , and) groups. Steady-state stimulation of Na⁺ transport withaldosterone was accompanied by a specific decrease of PKC proteinlevels in both the cytoplasmic and membrane fractions. Similarly,overnight treatment with an uncharged amiloride analog (CDPC), aprocedure that through feedback regulation causes a stimulation ofNa⁺ transport, also decreased PKC levels. These effectswere additive, indicating separate mechanisms that converge at thelevel of PKC. These effects were not accompanied by changes ofPKC mRNA levels as determined by Northern blot analysis. We proposethat this may represent a novel regulatory feedback mechanism necessary for sustaining an increase of Na⁺ transport.

     

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 caveolae in signal-transducing function of cardiac Na+/K+-ATPase

Ouabain binding toNa⁺/K⁺-ATPase activates Src/epidermal growthfactor receptor (EGFR) to initiate multiple signal pathways thatregulate growth. In cardiac myocytes and the intact heart, the earlyouabain-induced pathways that cause rapid activations of ERK1/2 alsoregulate intracellular Ca²⁺ concentration([Ca²⁺]_i) and contractility. The goal of thisstudy was to explore the role of caveolae in these early signalingevents. Subunits of Na⁺/K⁺-ATPase were detectedby immunoblot analysis in caveolae isolated from cardiac myocytes,cardiac ventricles, kidney cell lines, and kidney outer medulla byestablished detergent-free procedures. Isolated rat cardiac caveolaecontained Src, EGFR, ERK1/2, and 20-30% of cellular contents of₁- and ₂-isoforms ofNa⁺/K⁺-ATPase, along with nearly all ofcellular caveolin-3. Immunofluorescence microscopy of adult cardiacmyocytes showed the presence of caveolin-3 and -isoforms inperipheral sarcolemma and T tubules and suggested their partialcolocalization. Exposure of contracting isolated rat hearts to apositive inotropic dose of ouabain and analysis of isolated cardiaccaveolae showed that ouabain caused 1) no change in totalcaveolar ERK1/2, but a two- to threefold increase in caveolarphosphorylated/activated ERK1/2; 2) no change in caveolar ₁-isoform and caveolin-3; and 3) 50-60%increases in caveolar Src and ₂-isoform. These findings,in conjunction with previous observations, show that components of thepathways that link Na⁺/K⁺-ATPase to ERK1/2 and[Ca²⁺]_i are organized within cardiac caveolaemicrodomains. They also suggest that ouabain-induced recruitments ofSrc and ₂-isoform to caveolae are involved in themanifestation of the positive inotropic effect of ouabain.

     

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.

     

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.

     

Rabbit retinal neurons and glia express a variety of ENaC/DEG subunits

Some members of the epithelialNa⁺ channel/degenerin (ENaC/DEG) family of ion channelshave been detected in mammalian brain. Therefore, we examined the RNAand protein expression of these channels in another part of the centralnervous system, the rabbit retina. We next sought to demonstratephysiological evidence for an amiloride-sensitive current inMüller glia, which, on the basis of a previous study, are thoughtto express -ENaC (Golestaneh N, de Kozak Y, Klein C, and Mirshahi M. Glia 33: 160-168, 2001). RT-PCR of retinal RNA revealedthe presence of -, -, -, and -ENaC as well as acid-sensingion channel (ASIC)1, ASIC2, ASIC3, and ASIC4. Immunohistochemicallocalization with antibodies against -ENaC and -ENaC showedlabeling in Müller cells and neurons, respectively. The presenceof -ENaC, -ENaC, and ASIC1 was detected by Western blotting.Cultured Müller cells were whole cell patch clamped. These cellsexhibited an inward Na⁺ current that was blocked byamiloride. These data demonstrate for the first time both theexpression of a variety of ENaC and ASIC subunits in the rabbit retinaas well as distinct cellular expression patterns of specific subunitsin neurons and glia.

     

Regulation of the mitochondrial permeability transition by matrix Ca(2+) and voltage during anoxia/reoxygenation

Westudied the interplay between matrix Ca²⁺ concentration([Ca²⁺]) and mitochondrial membrane potential() in regulation of the mitochondrial permeability transition(MPT) during anoxia and reoxygenation. Without Ca²⁺loading, anoxia caused near-synchronous dissipation,mitochondrial Ca²⁺ efflux, and matrix volume shrinkage whena critically low PO₂ was reached, which wasrapidly reversible upon reoxygenation. These changes were related toelectron transport inhibition, not MPT. Cyclosporin A-sensitive MPT didoccur when extramitochondrial [Ca²⁺] was increased topromote significant Ca²⁺ uptake during anoxia, depending onthe Ca²⁺ load size and ability to maintain . However,when [Ca²⁺] was increased after complete dissipation, MPT did not occur until reoxygenation, at which timereactivation of electron transport led to partial regeneration.In the setting of elevated extramitochondrial Ca²⁺, thisenhanced matrix Ca²⁺ uptake while promoting MPT because ofless than full recovery of . The interplay between andmatrix [Ca²⁺] in accelerating or inhibiting MPT duringanoxia/reoxygenation has implications for preventing reoxygenationinjury associated with MPT.

     

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.

     

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.

     

PPAR-gamma ligands modulate effects of LPS in stimulated rat synovial fibroblasts

This work demonstrated the constitutive expressionof peroxisome proliferator-activated receptor (PPAR)- and PPAR-in rat synovial fibroblasts at both mRNA and protein levels. A decrease in PPAR- expression induced by 10 µg/ml lipopolysaccharide (LPS) was observed, whereas PPAR- mRNA expression was not modified. 15-Deoxy-^12,14-prostaglandin J₂(15d-PGJ₂) dose-dependently decreased LPS-induced cyclooxygenase (COX)-2 (80%) and inducible nitric oxide synthase (iNOS) mRNA expression (80%), whereas troglitazone (10 µM) only inhibited iNOS mRNA expression (50%). 15d-PGJ₂ decreasedLPS-induced interleukin (IL)-1 (25%) and tumor necrosis factor(TNF)- (40%) expression. Interestingly, troglitazone stronglydecreased TNF- expression (50%) but had no significant effect onIL-1 expression. 15d-PGJ₂ was able to inhibitDNA-binding activity of both nuclear factor (NF)-B and AP-1.Troglitazone had no effect on NF-B activation and was shown toincrease LPS-induced AP-1 activation. 15d-PGJ₂ andtroglitazone modulated the expression of LPS-induced iNOS, COX-2, andproinflammatory cytokines differently. Indeed, troglitazone seems tospecifically target TNF- and iNOS pathways. These results offer newinsights in regard to the anti-inflammatory potential of the PPAR-ligands and underline different mechanisms of action of15d-PGJ₂ and troglitazone in synovial fibroblasts.