Modulation of human neuronal alpha 1E-type calcium channel by alpha 2delta -subunit

Calcium channels are composed of a pore-forming subunit,₁, and at least two auxiliarysubunits, - and₂-subunits. It is well knownthat -subunits regulate most of the properties of the channel. Thefunction of ₂-subunit isless understood. In this study, the effects of the calcium channel₂-subunit on the neuronal_1E voltage-gated calciumchannel expressed in Xenopus oocyteswas investigated without and with simultaneous coexpression of eitherthe _1b- or the_2a-subunit. Most aspects of_1E function were affected by₂. Thus₂ caused a shift in thecurrent-voltage and conductance-voltage curves toward more positivepotentials and accelerated activation, deactivation, and theinstallation of the inactivation process. In addition, the efficiencywith which charge movement is coupled to pore opening assessed bydetermining ratios of limiting conductance to limiting charge movementwas decreased by ₂ byfactors that ranged from 1.6 (P < 0.01) for _1E-channels to 3.0 (P < 0.005) for_1E1b-channels. These results indicate that₂ facilitates the expressionand the maturation of_1E-channels and converts thesechannels into molecules responding more rapidly to voltage.

     

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

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

     

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.

     

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.

     

Intracellular H+ regulates the alpha -subunit of ENaC, the epithelial Na+ channel

Protons regulateelectrogenic sodium absorption in a variety of epithelia, including thecortical collecting duct, frog skin, and urinary bladder. Recently,three subunits (, , ) coding for the epithelial sodium channel(ENaC) were cloned. However, it is not known whether pH regulatesNa⁺ channels directly byinteracting with one of the three ENaC subunits or indirectly byinteracting with a regulatory protein. As a first step to identifyingthe molecular mechanisms of proton-mediated regulation of apicalmembrane Na⁺ permeability inepithelia, we examined the effect of pH on the biophysical propertiesof ENaC. To this end, we expressed various combinations of -, -,and -subunits of ENaC in Xenopusoocytes and studied ENaC currents by the two-electrode voltage-clampand patch-clamp techniques. In addition, the effect of pH on the-ENaC subunit was examined in planar lipid bilayers. We report that ,,-ENaC currents were regulated by changes in intracellular pH(pH_i) but not by changes inextracellular pH (pH_o).Acidification reduced and alkalization increased channel activity by avoltage-independent mechanism. Moreover, a reduction ofpH_i reduced single-channel openprobability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited ,-ENaC, ,-ENaC, and -ENaC currents. We conclude thatpH_i but notpH_o regulates ENaC and that the-ENaC subunit is regulated directly bypH_i.     

A second enzyme protecting mineralocorticoid receptors from glucocorticoid occupancy

We have confirmed that A6 cells (derived fromkidney of Xenopus laevis), whichcontain both mineralocorticoid and glucocorticoid receptors, do notnormally possess 11-hydroxysteroid dehydroxgenase (11-HSD1 or11-HSD2) enzymatic activity and so are without apparent "protective" enzymes. A6 cells do not convert the glucocorticoid corticosterone to 11-dehydrocorticosterone but do, however, possess steroid 6-hydroxylase that transforms corticosterone to6-hydroxycorticosterone. This hydroxylase is cytochromeP-450 3A (CYP3A). We have nowdetermined the effects of 3,5-tetrahydroprogesterone andchenodeoxycholic acid (both inhibitors of 11-HSD1) and11-dehydrocorticosterone and11-hydroxy-3,5-tetrahydroprogesterone (inhibitors of11-HSD2) and carbenoxalone, which inhibits both 11-HSD1 and11-HSD2, on the actions and metabolism of corticosterone and activeNa⁺ transport [short-circuitcurrent(I_sc)] inA6 cells. All of these 11-HSD inhibitory substances induced asignificant increment in corticosterone-inducedI_sc, which wasdetectable within 2 h. However, none of these agents caused an increasein I_sc whenincubated by themselves with A6 cells. In all cases, the additionalI_sc was inhibitedby the mineralocorticoid receptor (MR) antagonist, RU-28318, whereasthe original I_scelicited by corticosterone alone was inhibited by the glucocorticoidreceptor antagonist, RU-38486. In separate experiments, each agent wasshown to significantly inhibit metabolism of corticosterone to6-hydroxycorticosterone in A6 cells, and a linear relationshipexisted between 6-hydroxylase inhibition and the MR-mediatedincrease in I_scin the one inhibitor tested. Troleandomycin, a selective inhibitor ofCYP3A, inhibited 6-hydroxylase and also significantly enhancedcorticosterone-induced I_sc at 2 h. Theseexperiments indicate that the enhanced MR-mediated I_sc in A6 cellsmay be related to inhibition of 6-hydroxylase activity in thesecells and that this 6-hydroxylase (CYP3A) may be protecting theexpression of corticosterone-induced active Na⁺ transport in A6 cells byMR-mediated mechanism(s).

     

Colonic H-K-ATPase alpha- and beta-subunits express ouabain-insensitive H-K-ATPase

Active K absorption in the rat distal colon is energizedby an apical H-K-ATPase, a member of the gene family of P-type ATPases. The H-K-ATPase -subunit (HKc) has been cloned and characterized (together with the -subunit of either Na-K-ATPase or gastric H-K-ATPase) in Xenopus oocytes as ouabain-sensitive⁸⁶Rb uptake. In contrast, HKc, when expressed in Sf9cells without a -subunit, yielded evidence of ouabain-insensitiveH-K-ATPase. Because a -subunit (HKc) has recently been clonedfrom rat colon, this present study was initiated to determine whetherH-K-ATPase and its sensitivity to ouabain are expressed when these twosubunits (HKc and HKc) are transfected into a mammalian cellexpression system. Transfection of HEK-293 cells with HKc and HKccDNAs resulted in the expression of HKc and HKc proteins andtheir delivery to plasma membranes. H-K-ATPase activity was identified in crude plasma membranes prepared from transfected cells and was1) saturable as a function of increasing K concentration with aK_m for K of 0.63 mM; 2) inhibited byorthovanadate; and 3) insensitive to both ouabain andSch-28080. In parallel transfection studies with HKc and Na-K-ATPase1 cDNAs and with HKc cDNA alone, there was expression ofouabain-insensitive H-K-ATPase activity that was 60% and 21% of thatin HKc/HKc cDNA transfected cells, respectively. Ouabain-insensitive ⁸⁶Rb uptake was also identified incells transfected with HKc and HKc cDNAs. These studies establishthat HKc cDNA with HKc cDNA express ouabain-insensitiveH-K-ATPase similar to that identified in rat distal colon.

     

Bitter taste transduced by PLC-beta(2)-dependent rise in IP(3) and alpha-gustducin-dependent fall in cyclic nucleotides

Current evidence points to the existence of multiple processesfor bitter taste transduction. Previous work demonstrated involvement of the polyphosphoinositide system and an -gustducin(G_gust)-mediated stimulation of phosphodiesterase inbitter taste transduction. Additionally, a taste-enriched G protein-subunit, G₁₃, colocalizes with G_gustand mediates the denatonium-stimulated production of inositol1,4,5-trisphosphate (IP₃). Using quench-flow techniques, weshow here that the bitter stimuli, denatonium and strychnine, inducerapid (50-100 ms) and transient reductions in cAMP and cGMP andincreases in IP₃ in murine taste tissue. This decrease ofcyclic nucleotides is inhibited by G_gust antibodies,whereas the increase in IP₃ is not affected by antibodiesto G_gust. IP₃ production is inhibited byantibodies specific to phospholipase C-₂(PLC-₂), a PLC isoform known to be activated byG-subunits. Antibodies to PLC-₃ or toPLC-₄ were without effect. These data suggest atransduction mechanism for bitter taste involving the rapid andtransient metabolism of dual second messenger systems, both mediatedthrough a taste cell G protein, likely composed ofG_gust//₁₃, with both systems beingsimultaneously activated in the same bitter-sensitive taste receptor cell.

     

Heterologous desensitization of response mediated by selective PKC-dependent phosphorylation of G(i-1) and G(i-2)

This study examined the ability of protein kinase C (PKC) toinduce heterologous desensitization by targeting specific G proteinsand limiting their ability to transduce signals in smooth muscle.Activation of PKC by pretreatment of intestinal smooth muscle cellswith phorbol 12-myristate 13-acetate, cholecystokinin octapeptide, orthe phosphatase 1 and phosphatase 2A inhibitor, calyculin A,selectively phosphorylated G_i-1 and G_i-2,but not G_i-3 or G_o, and blockedinhibition of adenylyl cyclase mediated by somatostatin receptorscoupled to G_i-1 and opioid receptors coupled toG_i-2, but not by muscarinic M₂ and adenosineA₁ receptors coupled to G_i-3. Phosphorylationof G_i-1 and G_i-2 and blockade of cyclaseinhibition were reversed by calphostin C and bisindolylmaleimide, andadditively by selective inhibitors of PKC and PKC. Blockade ofinhibition was prevented by downregulation of PKC. Phosphorylation ofG-subunits by PKC also affected responses mediated by-subunits. Pretreatment of muscle cells withcANP-(4-23), a selective agonist of the natriureticpeptide clearance receptor, NPR-C, which activates phospholipase C(PLC)-3 via the -subunits of G_i-1 andG_i-2, inhibited the PLC- response to somatostatin and[D-Pen^2,5]enkephalin. The inhibition waspartly reversed by calphostin C. Short-term activation of PKC had noeffect on receptor binding or effector enzyme (adenylyl cyclase orPLC-) activity. We conclude that selective phosphorylation ofG_i-1 and G_i-2 by PKC partly accounts forheterologous desensitization of responses mediated by the - and-subunits of both G proteins. The desensitization reflects adecrease in reassociation and thus availability of heterotrimeric G proteins.

     

Interaction of alpha- and beta-subunits in native H-K-ATPase and cultured cells transfected with H-K-ATPase beta-subunit

The assembly of the -subunit of thegastric H-K-ATPase (HK) with the -subunit of the H-K-ATPase orthe Na-K-ATPase (NaK) was characterized with two anti-HKmonoclonal antibodies (MAbs). In fixed gastric oxyntic cells, inH-K-ATPase in vitro, and in Madin-Darby canine kidney (MDCK) cellstransfected with HK, MAb 2/2E6 was observed to bind to HK onlywhen interactions between - and -subunits were disrupted byvarious denaturants. The epitope for MAb 2/2E6 was mapped to thetetrapeptide S₂₂₆LHY₂₂₉ of the extracellulardomain of HK. The epitope for MAb 2G11 was mapped to the eightNH₂-terminal amino acids of the cytoplasmic domain ofHK. In transfected MDCK cells, MAb 2G11 could immunoprecipitate HK with -subunits of the endogenous cell surface NaK, as well as that from early in the biosynthetic pathway, whereas MAb 2/2E6 immunoprecipitated only a cohort of unassembled endoglycosidase H-sensitive HK. In HK-transfected LLC-PK₁ cells,significant immunofluorescent labeling of HK at the cell surfacecould be detected without postfixation denaturation or in live cells,although a fraction of transfected HK could also becoimmunoprecipitated with NaK. Thus assembly of HK with NaKdoes not appear to be a stringent requirement for cell surface deliveryof HK in LLC-PK₁ cells but may be required in MDCKcells. In addition, endogenous posttranslational regulatory mechanismsto prevent hybrid - heterodimer assembly appear to be compromisedin transfected cultured renal epithelial cells. Finally, theextracellular epitope for assembly-sensitive MAb 2/2E6 may represent aregion of HK that is associated with - interaction.

     

Unusual degradation of alpha -beta complexes in Xenopus oocytes by beta -subunits of Xenopus gastric H-K-ATPase

The catalytic -subunit of oligomeric P-type ATPases such asNa-K-ATPase and H-K-ATPase requires association with a -subunit after synthesis in the endoplasmic reticulum (ER) to become stably expressed and functionally active. In this study, we have expressed the-subunit of Xenopus gastricH-K-ATPase (HK) in Xenopus oocytes together with -subunits of H-K-ATPase (HK) or Na-K-ATPase (NK) and have followed the biosynthesis, assembly, and cell surface expression of functional pumps. Immunoprecipitations ofXenopus HK from metabolicallylabeled oocytes show that it is well expressed and, when synthesizedwithout -subunits, can leave the ER and become fully glycosylated.Xenopus HK can associate with both coexpressed HK and NK, but the - complexes formed aredegraded rapidly in or close to the ER and do not produce functionalpumps at the cell surface as assessed by⁸⁶Rb uptake. A possibleexplanation of these results is thatXenopus HK may contain atissue-specific signal that is important in the formation or correcttargeting of functional - complexes in the stomach but thatcannot be recognized in Xenopusoocytes and in consequence leads to cellular degradation of the -complexes in this experimental system.

     

Carbachol-induced desensitization of PLC-beta pathway in rat myometrium: downregulation of Gqalpha /G11alpha

In the estrogen-treated rat myometrium, carbachol increased thegeneration of inositol phosphates by stimulating the muscarinic receptor-G_q/G₁₁-phospholipaseC-3 (PLC-3) cascade. Exposure to carbachol resulted in a rapidand specific (homologous) attenuation of the subsequent muscarinicresponses in terms of inositol phosphate production, PLC-3translocation to membrane, and contraction. Refractoriness wasaccompanied by a reduction of membrane muscarinic binding sites and anuncoupled state of residual receptors. Protein kinase C (PKC) alteredthe functionality of muscarinic receptors and contributed to theinitial period of desensitization. A delayed phase of the muscarinicrefractoriness was PKC independent and was associated with adownregulation ofG_q/G₁₁.Atropine failed to induce desensitization as well asG_q/G₁₁downregulation, indicating that both events involve active occupancy ofthe receptor. Prolonged exposure toAlF₄ reduced subsequent AlF₄ as well as carbachol-mediatedinositol phosphate responses and similarly induced downregulation ofG_q/G₁₁. Data suggest that a decrease in the level ofG_q/G₁₁is subsequent to its activation and may account forheterologous desensitization.

     

Differential distribution of ERalpha and ERbeta mRNA in intrauterine tissues of the pregnant rhesus monkey

Twoestrogen receptor (ER) isoforms, ER and ER, have been described.However, no information is available in any species regarding thecomparison of ER and ER levels in pregnant intrauterine tissues.We investigated 1) distribution of ER and ER mRNA in myometrium, amnion, choriodecidua, and placenta; 2) theirabundance in intrauterine tissues at term not in labor (NIL) and inspontaneous term labor (STL); and 3) immunolocalization ofER and ER in pregnant rhesus monkey myometrium. Myometrium,amnion, choriodecidua, and placenta were obtained at cesarean sectionfrom monkeys in STL at 156-166 days gestational age(GA) (n = 4) and from control monkeys NIL at140-152 days GA (n = 4). RT-PCR was conducted to determineER and ER and glyceraldehyde-3-phosphate dehydrogenase mRNAabundance in four intrauterine tissues of the pregnant rhesus monkey.The cloned ER PCR fragment was subjected to sequence analysis. ERand ER were localized in the myometrium by immunohistochemistry. Wedemonstrated that 1) rhesus monkey ER shares >97%identity with human ER in the region sequenced; 2) both ERswere expressed in myometrium, amnion, and choriodecidua but not inplacenta in the current study; 3) ER and ER weredifferentially distributed in myometrium and amnion; 4) ERand ER were immunolocalized in myometrial smooth cells and smoothmuscle and endothelial cells of the myometrial blood vessels. Thebiological significance of these quantitative differences in ERsubtypes merits further study.

     

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.

     

alpha -Adrenergic receptors regulate human lymphocyte amiloride-sensitive sodium channels

Two independentsignal transduction pathways regulate lymphocyte amiloride-sensitivesodium channels (ASSCs), one utilizing cAMP as a second messenger andthe other utilizing a GTP-binding protein. This implies that two plasmamembrane receptors play a role in the regulation of lymphocyte ASSCs.In this study, we tested the hypothesis that₁- and₂-adrenergic receptorsindependently regulate lymphocyte ASSCs via the two previouslyidentified second messengers. Direct measurements indicated thatnorepinephrine increased lymphocyte cAMP and activated ASSCs. The₂-specific inhibitor,yohimbine, blocked this activation, thereby linking ₂-adrenergic receptors to ASSCregulation via cAMP. The₁-specific ligand, terazosin,acted as an agonist and activated lymphocyte ASSCs but inhibited ASSCcurrent that had been preactivated by norepinephrine or8-(4-chlorophenylthio) (CPT)-cAMP. Terazosin had no effect on thelymphocyte whole cell ASSC currents preactivated by treatment withpertussis toxin. This finding indirectly links ₁-adrenergic receptors tolymphocyte ASSC regulation via GTP-binding proteins. Terazosin had nodirect inhibitory or stimulatory effects on ,,-endothelialsodium channels reconstituted into planar lipid bilayers and expressedin Xenopus oocytes, ruling out a direct interaction between terazosin and the channels. These findings support the hypothesis that both₁- and₂-adrenergic receptors independently regulate lymphocyte ASSCs via GTP-binding proteins andcAMP, respectively.

     

Colonic H-K-ATPase beta -subunit: identification in apical membranes and regulation by dietary K depletion

P-type ATPasesrequire both - and -subunits for functionalactivity. Although an -subunit for colonic apical membraneH-K-ATPase (HKc) has been identified and studied, its -subunithas not been identified. We cloned putative -subunit rat colonicH-K-ATPase (HKc) cDNA that encodes a 279-amino-acid protein with asingle transmembrane domain and sequence homology to other rat-subunits. Northern blot analysis demonstrates that this HKc isexpressed in several rat tissues, including distal and proximal colon,and is highly expressed in testis and lung. HKc mRNA abundance is upregulated threefold compared with normal in distal colon but notproximal colon, testis, or lung of K-depleted rats. In contrast, Na-K-ATPase ₁ mRNA abundance isunaltered in distal colon of K-depleted rats. Na depletion, which alsostimulates active K absorption in distal colon, does not increaseHKc mRNA abundance. Western blot analyses using a polyclonalantibody raised to a glutathioneS-transferase-HKc fusion proteinestablished expression of a 45-kDa HKc protein in both apical andbasolateral membranes of rat distal colon, but K depletion increasedHKc protein expression only in apical membranes. Physicalassociation between HKc and HKc proteins was demonstrated byWestern blot analysis performed with HKc antibody onimmunoprecipitate of apical membranes of rat distal colon and HKcantibody. Tissue-specific upregulation of this -subunit mRNA inresponse to K depletion, localization of its protein, its upregulationby K depletion in apical membranes of distal colon, and its physicalassociation with HKc protein provide compelling evidence that HKcis the putative -subunit of colonic H-K-ATPase.     

TNF-alpha pretreatment prevents subsequent activation of cultured brain cells with TNF-alpha and hypoxia via ceramide

We havedeveloped a cellular model in which cultured astrocytes and braincapillary endothelial cells preconditioned with tumor necrosisfactor- (TNF-) fail to upregulate intercellular adhesionmolecule-1 (ICAM-1) protein (80% inhibition) and mRNA (30%inhibition) when challenged with TNF- or exposed to hypoxia. Inasmuch as ceramide is known to mediate some of the effects of TNF-, its levels were measured at various times after the TNF- preconditioning. We present evidence for the first time that, in normalbrain cells, TNF- pretreatment causes a biphasic increase ofceramide levels: an early peak at 15-20 min, when ceramide levelsincreased 1.9-fold in astrocytes and 2.7-fold in rat brain capillaryendothelial cells, and a delayed 2- to 3-fold ceramide increase thatoccurs 18-24 h after addition of TNF-. The following findingsindicate that the delayed ceramide accumulation results in cellunresponsiveness to TNF-: 1)coincident timing of the ceramide peak and the tolerance period,2) mimicking of preconditioning byaddition of exogenous ceramide, and3) attenuation of preconditioning byfumonisin B₁, an inhibitor ofceramide synthesis. In contrast to observations in transformed celllines, the delayed ceramide increase was transient and did not induceapoptosis in brain cells.     

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.

     

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.

     

Inhibition of cell differentiation by Galpha q in the renal epithelial cell line LLC-PK1

LLC-PK₁, an epithelial cellline derived from the kidney proximal tubule, was used to study theability of the G protein -subunit, G_q, to regulate celldifferentiation. A constitutively active mutant protein,_qQ209L, was expressed using theLacSwitch-inducible mammalian expression system. Induction of_qQ209L expression with isopropyl--D-thiogalactopyranoside(IPTG) enhanced phospholipase C activity maximally by 6- to 7.5-fold.Increasing concentrations of IPTG progressively inhibited the activityof two differentiation markers,Na⁺-dependent hexose transport andalkaline phosphatase activity. Induction of_qQ209L expression also caused achange from an epithelial to a spindle-shaped morphology. The effectsof _qQ209L expression on celldifferentiation were similar to those observed with12-O-tetradecanoylphorbol 13-acetate(TPA) treatment. However, protein kinase C (PKC) levels weredownregulated in TPA-treated cells but not in_qQ209L-expressing cells,suggesting that the regulation of PKC byG_q may be different fromregulation by TPA. Interestingly, the PKC inhibitor GF-109203X did notinhibit the effect of IPTG on the development ofNa⁺-dependent hexose transport in_qQ209L-expressing cells. These data implicate PKC and PKC in the pathway used byG_q to block the development ofNa⁺-dependent hexose transport inIPTG-treated cells.