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.

     

Osmotic pressure regulates alpha beta gamma -rENaC expressed in Xenopus oocytes

The hypothesisthat amiloride-sensitive Na⁺channels (ENaC) are involved in cell volume regulation was tested.Anisosmotic ND-20 media (ranging from 70 to 450 mosM) were used tosuperfuse Xenopus oocytes expressing-rat ENaC (-rENaC). Whole cell currents werereversibly dependent on external osmolarity. Under conditions ofswelling (70 mosM) or shrinkage (450 mosM), current amplitude decreasedand increased, respectively. In contrast, there was no change incurrent amplitude of H₂O-injectedoocytes to the above osmotic insults. Currents recorded from-rENaC-injected oocytes were not sensitive to externalCl concentration or to theK⁺ channel inhibitorBaCl₂. They were sensitive toamiloride. The concentration of amiloride necessary to inhibit one-halfof the maximal rENaC current expressed in oocytes(K_i; apparentdissociation constant) decreased in swollen cells and increased inshrunken oocytes. The osmotic pressure-inducedNa⁺ currents showed propertiessimilar to those of stretch-activated channels, including inhibition byGd³⁺ andLa³⁺, and decreased selectivityfor Na⁺.-rENaC-expressing oocytes maintained a nearly constant cell volume in hypertonic ND-20. The present study is the firstdemonstration that -rENaC heterologously expressed inXenopus oocytes may contribute tooocyte volume regulation following shrinkage.

     

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⁺.

     

IL-1beta-induced production of metalloproteinases by synovial cells depends on gap junction conductance

Synovial cells can form networksconnected by gap junctions. The purpose of this study was to obtainevidence for a necessary role of gap junction intercellularcommunication in protein secretion by synovial cells. We developed anovel assay to measure the enzymatic activity of metalloproteinases(MMPs) produced by synovial cells in response to interleukin-1(IL-1) and employed the assay to explore the biological function ofgap junctions. IL-1 produced a dose-dependent increase in MMPactivity that was blocked by exposure to the gap junction inhibitors18-glycyrrhetinic acid and octanol for as few as 50 min. Theinhibitors produced an immediate and marked reduction in intercellularcommunication, as assessed by transient current analysis using thenystatin perforated-patch method. These observations suggest thatcommunication through gap junctions early in IL-1 signaltransduction is critical to the process of cytokine-regulated secretionof MMPs by synovial cells.

     

PKA and arachidonic acid activation of human recombinant ClC-2 chloride channels

An HEK-293 cell line stably expressing the humanrecombinant ClC-2 Cl channel was used in patch-clampstudies to study its regulation. The relative permeabilityP_x/P_Cl calculated fromreversal potentials was I > Cl = NO₃ = SCNBr. Theabsolute permeability calculated from conductance ratios wasCl = Br = NO₃  SCN > I. The channel was activatedby cAMP-dependent protein kinase (PKA), reduced extracellular pH, oleicacid (C:18 cis9), elaidic acid (C:18trans9), arachidonic acid (AA; C:20cis5,8,11,14), and by inhibitors of AA metabolism,5,8,11,14-eicosatetraynoic acid (ETYA; C:20trans5,8,11,14),-methyl-4-(2-methylpropyl)benzeneacetic acid (ibuprofen), and2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2Cl channels were activated by a combination of forskolinplus IBMX and were inhibited by the cell-permeant myristoylated PKAinhibitor (mPKI). Channel activation by reduction of bath pH wasincreased by PKA and prevented by mPKI. AA activation of the ClC-2Cl channel was not inhibited by mPKI or staurosporine andwas therefore independent of PKA or protein kinase C activation.

     

Regulation of a cloned epithelial Na+ channel by its beta - and gamma -subunits

Using the Xenopus oocyteexpression system, we examined the mechanisms by which the - and-subunits of an epithelial Na⁺channel (ENaC) regulate -subunit channel activity and the mechanisms by which -subunit truncations cause ENaC activation. Expression of-ENaC alone produced small amiloride-sensitive currents (43 ± 10 nA, n = 7). These currentsincreased >30-fold with the coexpression of - and -ENaC to1,476 ± 254 nA (n = 20).This increase was accompanied by a 3.1- and 2.7-fold increase ofmembrane fluorescence intensity in the animal and vegetal poles of theoocyte, respectively, with use of an antibody directed against the-subunit of ENaC. Truncation of the last 75 amino acids of the-subunit COOH terminus, as found in the original pedigree ofindividuals with Liddle's syndrome, caused a 4.4-fold(n = 17) increase of theamiloride-sensitive currents compared with wild-type -ENaC.This was accompanied by a 35% increase of animal pole membranefluorescence intensity. Injection of a 30-amino acid peptide withsequence identity to the COOH terminus of the human -ENaCsignificantly reduced the amiloride-sensitive currents by 40-50%.These observations suggest a tonic inhibitory role on the channel'sopen probability (P_o) by the COOH terminus of -ENaC. We conclude that the changes of current observed with coexpression of the - and -subunits or those observed with -subunit truncation are likely the result ofchanges of channel density in combination with large changes ofP_o.

     

An immunologically distinct beta -adaptin on tubulovesicles of gastric oxyntic cells

Clathrin and the -adaptin subunit of the AP-1 clathrinadaptor have been previously identified on H-K-ATPase-richtubulovesicles from gastric acid secretory (oxyntic) cells [C. T. Okamoto, S. M. Karam, Y. Y. Jeng, J. G. Forte, and J. Goldenring.Am. J. Physiol. 274 (Cell Physiol. 43):C1017-C1029]. We further characterized this AP-1 adaptorfrom rabbit and hog tubulovesicles biochemically and immunologically.Clathrin coat proteins were stripped from purified tubulovesicularmembranes and fractionated by hydroxyapatite chromatography. The AP-1adaptor appears to elute at 200 mM sodium phosphate, based on thepresence of proteins in this fraction that are immunoreactive withantibodies against three of the four subunits of this heterotetramericcomplex: the -, µ₁-, and₁-adaptin subunits. Althoughthe putative -adaptin subunit in this fraction is not immunoreactivewith the anti--adaptin monoclonal antibody (MAb), this -adaptinis immunoreactive with polyclonal antibodies (PAbs) directed againstthe peptide sequenceGly⁶²⁵-Asp-Leu-Leu-Gly-Asp-Leu-Leu-Asn-Leu-Asp-Leu-Gly-Pro-Pro-Val⁶⁴⁰,a region conserved between ₁-and ₂-adaptins that is thought to be involved in the binding of clathrin heavy chain.Immunoprecipitation of the AP-1 adaptor complex from this fraction withanti--adaptin MAb 100/3 resulted in the coimmunoprecipitation of the-adaptin that did not react with the anti--adaptin MAb but didreact with the anti--adaptin PAbs. In contrast, immunoprecipitationof the AP-1 adaptor complex from crude clathrin-coated vesicles from brain resulted in the coimmunoprecipitation of a -adaptin that wasrecognized by both the anti--adaptin MAb and PAbs. These resultssuggest that the tubulovesicular AP-1 adaptor complex may be distinctfrom that found in the trans-Golgi network and may contain animmunologically distinct -adaptin. This immunologically distinct-adaptin may be diagnostic of apical tubulovesicular endosomes ofepithelial cells.

     

Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells

We investigated theeffects of epidermal growth factor (EGF) on activeNa⁺ absorption by alveolarepithelium. Rat alveolar epithelial cells (AEC) were isolated andcultivated in serum-free medium on tissue culture-treated polycarbonatefilters. mRNA for rat epithelial Na⁺ channel (rENaC) -, -,and -subunits and Na⁺ pump₁- and₁-subunits were detected inday 4 monolayers by Northern analysisand were unchanged in abundance in day5 monolayers in the absence of EGF. Monolayerscultivated in the presence of EGF (20 ng/ml) for 24 h fromday 4 to day5 showed an increase in both₁ and₁Na⁺ pump subunit mRNA but noincrease in rENaC subunit mRNA. EGF-treated monolayers showed parallelincreases in Na⁺ pump₁- and₁-subunit protein by immunoblotrelative to untreated monolayers. Fixed AEC monolayers demonstratedpredominantly membrane-associated immunofluorescent labeling withanti-Na⁺ pump₁- and₁-subunit antibodies, withincreased intensity of cell labeling for both subunits seen at 24 hfollowing exposure to EGF. These changes inNa⁺ pump mRNA and protein precededa delayed (>12 h) increase in short-current circuit (measure ofactive transepithelial Na⁺transport) across monolayers treated with EGF compared with untreated monolayers. We conclude that EGF increases activeNa⁺ resorption across AECmonolayers primarily via direct effects onNa⁺ pump subunit mRNA expressionand protein synthesis, leading to increased numbers of functionalNa⁺ pumps in the basolateralmembranes.

     

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.

     

Synthesis and characterization of dual-wavelength Cl--sensitive fluorescent indicators for ratio imaging

The fluorescence of quinolinium-basedCl indicators such as6-methoxy-N-(3-sulfopropyl)quinolinium(SPQ) is quenched by Cl bya collisional mechanism without change in spectral shape. A series of"chimeric" dual-wavelengthCl indicators weresynthesized by conjugatingCl-sensitive and-insensitive chromophores with spacers. The SPQ chromophore(N-substituted 6-methoxyquinolinium; MQ) was selected as theCl-sensitive moiety[excitation wavelength(_ex) 350 nm, emission wavelength (_em) 450 nm]. N-substituted 6-aminoquinolinium (AQ) waschosen as theCl-insensitive moietybecause of its different spectral characteristics (_ex 380 nm,_em 546 nm), insensitivity toCl, positive charge (tominimize quenching by chromophore stacking/electron transfer), andreducibility (for noninvasive cell loading). The dual-wavelengthindicators were stable and nontoxic in cells and were distributeduniformly in cytoplasm, with occasional staining of the nucleus. Thebrightest and mostCl-sensitive indicatorswere -MQ-'-dimethyl-AQ-xylene dichloride andtrans-1,2-bis(4-[1-'-MQ-1'-'-dimethyl-AQ-xylyl]-pyridinium)ethylene (bis-DMXPQ). At 365-nm excitation, emission maxima were at 450 nm(Cl sensitive; Stern-Volmerconstants 82 and 98 M¹)and 565 nm (Clinsensitive). Cystic fibrosis transmembrane conductanceregulator-expressing Swiss 3T3 fibroblasts were labeled with bis-DMXPQby hypotonic shock or were labeled with its uncharged reduced form(octahydro-bis-DMXPQ) by brief incubation (20 µM, 10 min). Changes inCl concentration inresponse to Cl/nitrateexchange were recorded by emission ratio imaging (450/565 nm) at 365-nmexcitation wavelength. These results establish a first-generation setof chimeric bisquinoliniumCl indicators forratiometric measurement ofCl concentration.     

Upregulation of Kv1.3 K(+) channels in microglia deactivated by TGF-beta

Microglial activation is accompanied by changes inK⁺ channel expression. Here we demonstrate that adeactivating cytokine changes the electrophysiological properties ofmicroglial cells. Upregulation of delayed rectifier (DR) K⁺channels was observed in microglia after exposure to transforming growth factor- (TGF-) for 24 h. In contrast, inwardrectifier K⁺ channel expression was unchanged by TGF-.DR current density was more than sixfold larger in TGF--treatedmicroglia than in untreated microglia. DR currents of TGF--treatedcells exhibited the following properties: activation at potentials morepositive than 40 mV, half-maximal activation at 27 mV, half-maximalinactivation at 38 mV, time dependent and strongly use-dependentinactivation, and a single channel conductance of 13 pS in Ringersolution. DR channels were highly sensitive to charybdotoxin (CTX) andkaliotoxin (KTX), whereas -dendrotoxin had little effect.With RT-PCR, mRNA for Kv1.3 and Kir2.1 was detected in microglia. Inaccordance with the observed changes in DR current density, the mRNAlevel for Kv1.3 (assessed by competitive RT-PCR) increased fivefold after treatment of microglia with TGF-.

     

AVP V1 receptor-mediated decrease in Cl- efflux and increase in dark cell number in choroid plexus epithelium

The cerebrospinalfluid (CSF)-generating choroid plexus (CP) has manyV₁ binding sites for argininevasopressin (AVP). AVP decreases CSF formation rate and choroidal bloodflow, but little is known about how AVP alters ion transport across theblood-CSF barrier. Adult rat lateral ventricle CP was loaded with³⁶Cl,exposed to AVP for 20 min, and then placed in isotope-free artificial CSF to measure release of³⁶Cl.Effect of AVP at 10¹² to10⁷ M on theCl efflux rate coefficient(in s¹) was quantified.Maximal inhibition (by 20%) ofCl extrusion at10⁹ M AVP was prevented bythe V₁ receptor antagonist[-mercapto-,-cyclopentamethyleneproprionyl¹,O-Me-Tyr²,Arg⁸]vasopressin.AVP also increased by more than twofold the number of dark and possiblydehydrated but otherwise morphologically normal choroid epithelialcells in adult CP. The V₁ receptorantagonist prevented this AVP-induced increment in dark cell frequency.In infant rats (1 wk) with incomplete CSF secretory ability,10⁹ M AVP altered neitherCl efflux nor dark cellfrequency. The ability of AVP to elicit functional and structuralchanges in adult, but not infant, CP epithelium is discussed in regardto ion transport, CSF secretion, intracranial pressure, and hydrocephalus.

     

Intracellular signaling mechanisms of interleukin-1beta in synovial fibroblasts

The possibility that membrane depolarization of synovialfibroblasts caused by interleukin-1 (IL-1) was mediated byprotein kinase C (PKC) and Ca²⁺influx was studied using inhibitor and activator analysis. The effectof IL-1 was blocked by bisindolylmaleimide I, an inhibitor of PKC,and by the Ca²⁺ channel blockersnifedipine and verapamil. In other experiments, PKC was activated usingphorbol 12-myristate 13-acetate, andCa²⁺ influx was increased by meansof a Ca²⁺ ionophore. Simultaneousapplication of phorbol ester andCa²⁺ ionophore in the absence ofIL-1 mimicked the depolarization caused by IL-1. The results wereconsistent with the hypothesis that, under the conditions studied,activation of PKC and Ca²⁺ influxare necessary and sufficient processes in the transduction of IL-1by synovial cells leading to membrane depolarization. Theessential role of protein phosphorylation andCa²⁺ influx in the earlyelectrophysiological response of synovial fibroblasts to IL-1 wastherefore established. The role of IL-1-induced depolarization inregulating protein expression by the cells remains to be determined,but the results reported here, taken together with observations thatprotein phosphorylation and Ca²⁺influx also mediate the effect of IL-1 on protease production (1, 2), suggest that electrophysiological changes are actually part of thepathway for expression of proteases in response to IL-1.

     

Peroxynitrite inhibits amiloride-sensitive Na+ currents in Xenopus oocytes expressing alpha beta gamma -rENaC

We examined the effect of peroxynitrite(ONOO) on the cloned ratepithelial Na⁺ channel(-rENaC) expressed in Xenopusoocytes. 3-Morpholinosydnonimine (SIN-1) was used to concurrentlygenerate nitric oxide (· NO) and superoxide(O₂ ·), which react toform ONOO, a species knownto promote protein nitration and oxidation. Under control conditions,oocytes displayed an amiloride-sensitive whole cell conductance of 7.4 ± 2.8 (SE) µS. When incubated at 18°C with SIN-1 (1 mM) for 2 h (final ONOO concentration = 10 µM), the amiloride-sensitive conductance was reduced to0.8 ± 0.5 µS. To evaluate whether the observed inhibition was due to ONOO, as opposedto · NO, we also exposed oocytes to SIN-1 in the presence ofurate (500 µM), a scavenger ofONOO and superoxidedismutase, which scavengesO₂ ·, converting SIN-1from an ONOO to an· NO donor. Under these conditions, conductance values remained at control levels following SIN-1 treatment.Tetranitromethane, an agent that oxidizes sulfhydryl groups at pH6, also inhibited the amiloride-sensitive conductance. These datasuggest that oxidation of critical sulfhydryl groups within rENaC byONOO directly inhibitschannel activity.

     

KGF prevents hyperoxia-induced reduction of active ion transport in alveolar epithelial cells

We evaluated theeffects of acute hyperoxic exposure on alveolar epithelial cell (AEC)active ion transport and on expression ofNa⁺ pump(Na⁺-K⁺-ATPase)and rat epithelial Na⁺ channelsubunits. Rat AEC were cultivated in minimal defined serum-free medium(MDSF) on polycarbonate filters. Beginning on day5, confluent monolayers were exposedto either 95% air-5% CO₂(normoxia) or 95% O₂-5%CO₂ (hyperoxia) for 48 h.Transepithelial resistance(R_t) andshort-circuit current(I_sc) weredetermined before and after exposure.Na⁺ channel -, -, and-subunit andNa⁺-K⁺-ATPase₁- and₁-subunit mRNA levels werequantified by Northern analysis.Na⁺ pump₁- and₁-subunit protein abundance wasquantified by Western blotting. After hyperoxic exposure,I_sc across AECmonolayers decreased by ~60% at 48 h relative to monolayersmaintained under normoxic conditions.Na⁺ channel -subunit mRNAexpression was reduced by hyperoxia, whereas - and -subunit mRNAexpression was unchanged. Na⁺ pump₁-subunit mRNA was unchanged,whereas ₁-subunit mRNA was decreased ~80% by hyperoxia in parallel with a reduction in₁-subunit protein. Becausekeratinocyte growth factor (KGF) has recently been shown to upregulateAEC active ion transport and expression ofNa⁺-K⁺-ATPaseunder normoxic conditions, we assessed the ability of KGF to preventhyperoxia-induced changes in active ion transport by supplementingmedium with KGF (10 ng/ml) from day2. The presence of KGF prevented theeffects of hyperoxia on ion transport (as measured byI_sc) relativeto normoxic controls. Levels of₁ mRNA and protein wererelatively preserved in monolayers maintained in MDSF and KGF comparedwith those cultivated in MDSF alone. These results indicate that AECnet active ion transport is decreased after 48 h of hyperoxia, likelyas a result of a decrease in the number of functionalNa⁺ pumps per cell. KGF largelyprevents this decrease in active ion transport, at least in part, bypreserving Na⁺ pump expression.

     

Overexpression of protein kinase C-delta increases tight junction permeability in LLC-PK1 epithelia

The Ca²⁺-independent-isoform of protein kinase C (PKC-) was overexpressed inLLC-PK₁ epithelia and placed undercontrol of a tetracycline-responsive expression system. In the absenceof tetracycline, the exogenous PKC- is expressed. Westernimmunoblots show that the overexpressed PKC- is found in thecytosolic, membrane-associated, and Triton-insoluble fractions.Overexpression of PKC- produced subconfluent and confluentepithelial morphologies similar to that observed on exposure ofwild-type cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Transepithelialelectrical resistance(R_T) in cellsheets overexpressing PKC- was only 20% of that in cell sheetsincubated in the presence of tetracycline, in which the amount ofPKC- and R_Twere similar to those in LLC-PK₁ parental cell sheets. Overexpression of PKC- also elicited a significant increase in transepithelial flux ofD-[¹⁴C]mannitoland a radiolabeled 2 × 10⁶-molecular-weight dextran,suggesting with theR_T decrease that overexpression increased paracellular, tight junctional permeability. Electron microscopy showed that PKC- overexpression results in amultilayered cell sheet, the tight junctions of which are almost uniformly permeable to ruthenium red. Freeze-fracture electron microscopy indicates that overexpression of PKC- results in a moredisorganized arrangement of tight junctional strands. As withLLC-PK₁ cell sheets treated with12-O-tetradecanoylphorbol-13-acetate, the reducedR_T, increasedD-mannitol flux, and tightjunctional leakiness to ruthenium red that are seen with PKC-overexpression suggest the involvement of PKC- in regulation oftight junctional permeability.

     

High glucose and endothelial cell growth: novel effects independent of autocrine TGF-beta 1 and hyperosmolarity

Human endothelial cells wereexposed to 5 mM glucose (control), 25 mM (high) glucose, or osmoticcontrol for 72 h. TGF-1 production, cell growth, death, andcell cycle progression, and the effects of TGF-1 and TGF-neutralization on these parameters were studied. High glucose andhyperosmolarity increased endothelial TGF-1 secretion(P < 0.0001) and bioactivity (P < 0.0001). However, high glucose had a greater effect on reducingendothelial cell number (P < 0.001) and increasingcellular protein content (P < 0.001) than the osmoticcontrol. TGF- antibody only reversed the antiproliferative andhypertrophic effects of high glucose. High glucose altered cell cycleprogression and cyclin-dependent kinase inhibitor expressionindependently of hyperosmolarity. High glucose increased endothelialcell apoptosis (P < 0.01), whereashyperosmolarity induced endothelial cell necrosis (P < 0.001). TGF- antibody did not reverse the apoptotic effectsobserved with high glucose. Exogenous TGF-1 mimicked the increased Sphase delay but not endoreduplication observed with high glucose. High glucose altered endothelial cell growth, apoptosis, and cellcycle progression. These growth effects occurred principally via aTGF-1 autocrine pathway. In contrast, apoptosis andendoreduplication occurred independently of this cytokine and hyperosmolarity.

     

Time-dependent changes in myosin heavy chain mRNA and protein isoforms in unloaded soleus muscle of rat

Time-dependent changes in myosin heavy chain(MHC) isoform expression were investigated in rat soleus muscleunloaded by hindlimb suspension. Changes at the mRNA level weremeasured by RT-PCR and correlated with changes in the pattern of MHCprotein isoforms. Protein analyses of whole muscle revealed that MHCIdecreased after 7 days, when MHCIIa had increased, reaching a transient maximum by 15 days. Longer periods led to inductions and progressive increases of MHCIId(x) and MHCIIb. mRNA analyses of whole muscle showedthat MHCIId(x) displayed the steepest increase after 4 days andcontinued to rise until 28 days, the longest time period investigated.MHCIIb mRNA followed a similar time course, although at lower levels.MHCI mRNA, present at extremely low levels in control soleus, peakedafter 4 days, stayed elevated until 15 days, and then decayed.Immunohistochemistry of 15-day unloaded muscles revealed that MHCIwas present in muscle spindles but at low amounts also in extrafusalfibers. The slow-to-fast transitions thus seem to proceed in the orderMHCI  MHCIIa  MHCIId(x)  MHCIIb. Ourfindings indicate that MHCI is transiently upregulated in somefibers as an intermediate step during the transition from MHCI to MHCIIa.

     

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.

     

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.