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.

     

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.

     

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.

     

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.     

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.

     

Activation of p42mapk in human umbilical vein endothelial cells by interleukin-1alpha and tumor necrosis factor-alpha

Work from this and other laboratories has identified a role forprotein tyrosine kinases in interleukin-1 (IL-1)- and tumor necrosis factor- (TNF-)-induced responses in endothelial cells. In this study, we show that activation of human umbilical vein endothelial cells (HUVEC) by IL-1 leads to increased tyrosine phosphorylation of several proteins including one with a molecular massof ~42 kDa. This protein was identified asp42^mapk by Western blot analysis.Tyrosine phosphorylation and catalytic activation ofp42^mapk by IL-1 was transient,reaching maximal levels after 30 min and returning to basal levels by120-300 min. Activation ofp42^mapk in HUVEC was also observedin response to TNF- or to the protein kinase C (PKC)-activatingphorbol ester phorbol 12-myristate 13-acetate (PMA). Pretreatment ofHUVEC with IL-1 or TNF- prevented reactivation ofp42^mapk by either cytokine but didnot affect subsequent activation in response to PMA. Activation ofp42^mapk by PMA was significantlyreduced by the PKC inhibitor Ro-31-8220 and completely inhibited by theprotein tyrosine kinase inhibitor genistein. Genistein, but notRo-31-8220, attenuated IL-1- and TNF--inducedp42^mapk activation. Takentogether, the results of this study demonstrate 1) thatp42^mapk is transiently activatedin HUVEC by IL-1 and TNF-, 2)that this activation is PKC independent, and3) that a genistein-inhibitable tyrosine kinase may be an upstream regulator of cytokine-induced p42^mapk activation in humanendothelium.

     

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.

     

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.

     

Cloning and functional studies of splice variants of the alpha -subunit of the amiloride-sensitive Na+ channel

The -subunit of the amiloride-sensitive epithelialNa⁺ channel (ENaC) is criticalin forming an ion conductive pore in the membrane. We have identifiedthe wild-type and three splice variants of the human ENaC (hENaC)from the human lung cell line H441, using RT-PCR. These splice variantscontain various structures in the extracellular domain, resultingin premature truncation (hENaCx), 19-amino acid deletion(hENaC19), and 22-amino acid insertion (hENaC+22).Wild-type hENaC and splice variants were functionally characterizedin Xenopus oocytes by coexpression with hENaC - and -subunits. Unlike wild-type hENaC,undetectable or substantially reduced amiloride-sensitive currents wereobserved in oocytes expressing these splice variants. Wild-typehENaC was the most abundantly expressed hENaC mRNA species in alltissues in which its expression was detected. These findings indicate that the extracellular domain is important to generate structural andfunctional diversity of hENaC and that alternative splicing may playa role in regulating hENaC activity.

     

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.     

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.     

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.

     

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.

     

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.

     

Effect of TNF-alpha on SMIT mRNA levels and myo-inositol accumulation in cultured endothelial cells

Previously we have shown that hyperosmolarity increasesNa⁺-myo-inositolcotransporter (SMIT) activity and mRNA levels in cultured endothelialcells. Because hyperosmolarity and cytokines, such as tumor necrosisfactor- (TNF-), activate similar signal transduction pathways, weexamined the effect of TNF- on SMIT mRNA levels andmyo-inositol accumulation. In contrastto the effect of hyperosmolarity, TNF- caused a time- andconcentration-dependent decrease in SMIT mRNA levels andmyo-inositol accumulation. The effectof TNF- on myo-inositolaccumulation was found in large-vessel endothelial cells (derived fromthe aorta and pulmonary artery) and cerebral microvessel endothelialcells. In bovine aorta and bovine pulmonary artery endothelial cells,TNF- activated nuclear factor (NF)-B. TNF- also increasedceramide levels, and C₂-ceramidemimicked the effect of TNF- on SMIT mRNA levels andmyo-inositol accumulation in bovineaorta endothelial cells. Pyrrolidinedithiocarbamate, genistein, and7-amino-1-chloro-3-tosylamido-2-hepatanone, compounds that can inhibitNF-B activation, partially prevented the TNF--induced decrease inmyo-inositol accumulation. The effectof TNF- on myo-inositolaccumulation was also partially prevented by the protein kinase Cinhibitor calphostin C but not by staurosporine. These studiesdemonstrate that TNF- causes a decrease in SMIT mRNA levels andmyo-inositol accumulation in culturedendothelial cells, which may be related to the activation of NF-B.

     

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.

     

beta-adrenergic receptor/cAMP-mediated signaling and apoptosis of S49 lymphoma cells

-Adrenergic receptor (AR) activationand/or increases in cAMP regulate growth and proliferation of a varietyof cells and, in some cells, promote cell death. In the current studieswe addressed the mechanism of this growth reduction by examiningAR-mediated effects in the murine T-lymphoma cell line S49.Wild-type S49 cells, derived from immature thymocytes(CD4⁺/CD8⁺) undergo growth arrest andsubsequent death when treated with agents that increase cAMP levels(e.g., AR agonists, 8-bromo-cAMP, cholera toxin, forskolin).Morphological and biochemical criteria indicate that this cell death isa result of apoptosis. In cyc and kin S49cells, which lack G_s and functional protein kinase A(PKA), respectively, AR activation of G_s and cAMPaction via PKA are critical steps in this apoptotic pathway. S49 cellsthat overexpress Bcl-2 are resistant to cAMP-induced apoptosis. Weconclude that AR activation induces apoptosis in immature Tlymphocytes via G_s and PKA, while overexpression ofBcl-2 prevents cell death. AR/cAMP/PKA-mediated apoptosis mayprovide a means to control proliferation of immature T cells in vivo.

     

Nitric oxide-dependent downregulation of adipocyte UCP-2 expression by tumor necrosis factor-alpha

Uncoupling protein-2 (UCP-2) is amitochondrial protein expressed in adipocytes and has recently beeninvolved in the control of energy dissipation. Because obesity ischaracterized by an imbalance between energy intake and expenditure andby an enhanced adipocyte-derived secretion of tumor necrosis factor-(TNF-), we asked whether TNF- could directly influence UCP-2expression in adipocytes. Experiments performed in differentiated3T3F442A preadipocytes showed that TNF- (10 ng/ml) induced areduction of UCP-2 trancripts, assessed by Northern blot analysis. Asignificant decrease in UCP-2 expression (40%) was observed after 12 and 24 h of TNF- stimulation of the cells. The characterizationof the mechanisms responsible for the TNF- effect on UCP-2expression demonstrates an involvement of the TNF--induced inducible(i) nitric oxide synthase (NOS) expression. Cell treatment with the NOSinhibitor N^G-nitro-L-arginine methylester (L-NAME; 1 mmol/l) significantly diminished theTNF--mediated sustained downregulation of UCP-2 expression, whereascell treatment with a nitric oxide (NO) donor (10³ mol/lS-nitroso-L-glutathione) mimicked the TNF-effect on UCP-2 expression. Moreover, Western blot analysis clearlyshowed that TNF- alone induces the expression of iNOS after12-24 h treatment of differentiated 3T3F442A cells. Theseexperiments demonstrate that TNF- directly downregulates UCP-2expression via NO-dependent pathways that involve the induction of iNOS expression.

     

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.

     

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).