Regulation of the cardiac L-type Ca2+ channel by the actin-binding proteins alpha-actinin and dystrophin

Theactin-binding proteins dystrophin and -actinin are members of afamily of actin-binding proteins that may link the cytoskeleton tomembrane proteins such as ion channels. Previous work demonstrated thatthe activity of Ca²⁺ channels can be regulated by agentsthat disrupt or stabilize the cytoskeleton. In the present study, weemployed immunohistochemical and electrophysiological techniques toinvestigate the potential regulation of cardiac L-type Ca²⁺channel activity by dystrophin and -actinin in cardiac myocytes andin heterologous cells. Both actin-binding proteins were found tocolocalize with the Ca²⁺ channel in mouse cardiac myocytesand to modulate channel function. Inactivation of the Ca²⁺channel in cardiac myocytes from mice lacking dystrophin(mdx mice) was reduced compared with that in wild-typemyocytes, voltage dependence of activation was shifted by 5 mV to morepositive potentials, and stimulation by the -adrenergic pathway andthe dihydropyridine agonist BAY K 8644 was increased. Furthermore, heterologous coexpression of the Ca²⁺ channel with muscle,but not nonmuscle, forms of -actinin was also found to reduceinactivation. As might be predicted from a reduction ofCa²⁺ channel inactivation, a prolonging of the mouseelectrocardiogram QT was observed in mdx mice. These resultssuggest a combined role for dystrophin and -actinin in regulatingthe activity of the cardiac L-type Ca²⁺ channel and apotential mechanism for cardiac dysfunction in Duchenne and Beckermuscular dystrophies.

     

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.

     

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.

     

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.

     

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.

     

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.

     

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-.

     

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.

     

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.

     

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.

     

Insulin increases plasma membrane content and reduces phosphorylation of Na+-K+ pump alpha 1-subunit in HEK-293 cells

Insulin stimulates K⁺ uptake andNa⁺ efflux via the Na⁺-K⁺ pump inkidney, skeletal muscle, and brain. The mechanism of insulin action inthese tissues differs, in part, because of differences in the isoformcomplement of the catalytic -subunit of theNa⁺-K⁺ pump. To analyze specifically the effectof insulin on the ₁-isoform of the pump, we have studiedhuman embryonic kidney (HEK)-293 cells stably transfected with the ratNa⁺-K⁺ pump ₁-isoform tagged onits first exofacial loop with a hemagglutinin (HA) epitope. The plasmamembrane content of ₁-subunits was quantitated bybinding a specific HA antibody to intact cells. Insulin rapidly increased the number of ₁-subunits at the cell surface.This gain was sensitive to the phosphatidylinositol (PI) 3-kinaseinhibitor wortmannin and to the protein kinase C (PKC) inhibitorbisindolylmaleimide. Furthermore, the insulin-stimulated gain insurface -subunits correlated with an increase in the binding of anantibody that recognizes only the nonphosphorylated form of₁ (at serine-18). These results suggest that insulinregulates the Na⁺-K⁺ pump in HEK-293 cells, atleast in part, by decreasing serine phosphorylation and increasingplasma membrane content of ₁-subunits via a signalingpathway involving PI 3-kinase and PKC.

     

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.

     

Stability of actin cytoskeleton and PKC-delta binding to actin regulate NKCC1 function in airway epithelial cells

Activation of airwayepithelial Na-K-2Cl cotransporter (NKCC)1 requires increased activityof protein kinase C (PKC)-, which localizes predominantly to theactin cytoskeleton. Prompted by reports of a role for actin in NKCC1function, we studied a signaling mechanism linking NKCC1 and PKC.Stabilization of actin polymerization with jasplakinolide increasedactivity of NKCC1, whereas inhibition of actin polymerization withlatrunculin B prevented hormonal activation of NKCC1. Protein-proteininteractions among NKCC1, actin, and PKC- were verified by Westernblot analysis of immunoprecipitated proteins. PKC- was detected inimmunoprecipitates of NKCC1 and vice versa. Actin was also detected inimmunoprecipitates of NKCC1 and PKC-. Pulldown of endogenous actinrevealed the presence of NKCC1 and PKC-. Binding of recombinantPKC- to NKCC1 was not detected in overlay assays. Rather, activatedPKC- bound to actin, and this interaction was prevented by a peptideencoding C2, a C2-like domain based on the amino acid sequence ofPKC-. C2 also blocked stimulation of NKCC1 function bymethoxamine. Immunofluorescence and confocal microscopy revealedPKC- in the cytosol and cell periphery. Merged images of cellsstained for actin and PKC- indicated colocalization of PKC- andactin at the cell periphery. The results indicate that actin iscritical for the activation of NKCC1 through a direct interaction with PKC-.

     

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.

     

Regulation of Fas (CD95)-induced apoptosis by nuclear factor-kappaB and tumor necrosis factor-alpha in macrophages

The APO-1/Fasligand (FasL) and tumor necrosis factor- (TNF-) are twofunctionally related molecules that induce apoptosis ofsusceptible cells. Although the two molecules have been reported toinduce apoptosis via distinct signaling pathways, we have shown that FasL can also upregulate the expression of TNF-, raising thepossibility that TNF- may be involved in FasL-inducedapoptosis. Because TNF- gene expression is under the controlof nuclear factor-B (NF-B), we investigated whether FasL caninduce NF-B activation and whether such activation plays a role inFasL-mediated cell death in macrophages. Gene transfection studiesusing NF-B-dependent reporter plasmid showed that FasL did activateNF-B promoter activity. Gel shift studies also revealed that FasLmobilized the p50/p65 heterodimeric form of NF-B. Inhibition ofNF-B by a specific NF-B inhibitor, caffeic acid phenylethylester, or by dominant expression of the NF-B inhibitory subunitIB caused an increase in FasL-induced apoptosis and areduction in TNF- expression. However, neutralization of TNF- byspecific anti-TNF- antibody had no effect on FasL-inducedapoptosis. These results indicate that FasL-mediated cell deathin macrophages is regulated through NF-B and is independent ofTNF- activation, suggesting the antiapoptotic role of NF-Band a separate death signaling pathway mediated by FasL.

     

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.

     

Combined modulation of the mesangial machinery for monocyte recruitment by inhibition of NF-kappa B

The activation of nuclear factor-B(NF-B) is required for the induction of many of the adhesionmolecules and chemokines involved in the inflammatory leukocyterecruitment to the kidney. Here we studied the effects of NF-Binhibition on the machinery crucial for monocyte infiltration of theglomerulus during inflammation. In mesangial cells (MC), the proteaseinhibitors MG-132 and N--tosyl-L-lysine chloromethyl ketone or adenoviral overexpression of IB- prevented the complete IB- degradation following tumor necrosis factor- (TNF-) stimulation. This resulted in a marked inhibition ofTNF--induced expression of mRNA and protein for the immunoglobulinmolecules intracellular adhesion molecule-1 and vascular cell adhesionmolecule-1 and the chemokines growth-related oncogene-, monocytechemoattractant protein-1, interleukin-8, or fractalkine in MC.Finally, the inhibition of IB- degradation or IB-overexpression suppressed the chemokine-induced transendothelialmonocyte chemotaxis toward MC and the chemokine-triggered firm adhesionof monocytic cells to MC. The inhibition of NF-B by pharmacologicalintervention or gene transfer may present a multimodal approach tocontrol the machinery propagating inflammatory recruitment of monocytesduring glomerular disease.

     

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 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.