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.

     

NF-kappa B inactivation converts a hepatocyte cell line TNF-alpha response from proliferation to apoptosis

Toxins convertthe hepatocellular response to tumor necrosis factor- (TNF-)stimulation from proliferation to cell death, suggesting thathepatotoxins somehow sensitize hepatocytes to TNF- toxicity. Becausenuclear factor-B (NF-B) activation confers resistance to TNF-cytotoxicity in nonhepatic cells, the possibility that toxin-inducedsensitization to TNF- killing results from inhibition ofNF-B-dependent gene expression was examined in the RALA rathepatocyte cell line sensitized to TNF- cytotoxicity by actinomycinD (ActD). ActD did not affect TNF--induced hepatocyte NF-Bactivation but decreased NF-B-dependent gene expression. Expressionof an IB superrepressor rendered RALA hepatocytes sensitive toTNF--induced apoptosis in the absence of ActD. Apoptosis was blockedby caspase inhibitors, and TNF- treatment led to activation ofcaspase-2, caspase-3, and caspase-8 only when NF-B activation wasblocked. Although apoptosis was blocked by the NF-B-dependent factornitric oxide (NO), inhibition of endogenous NO production did notsensitize cells to TNF--induced cytotoxicity. Thus NF-Bactivation is the critical intracellular signal that determines whetherTNF- stimulates hepatocyte proliferation or apoptosis. Althoughexogenous NO blocks RALA hepatocyte TNF- cytotoxicity, endogenousproduction of NO is not the mechanism by which NF-B activationinhibits this death pathway.

     

CCK independently activates intracellular trypsinogen and NF-kappaB in rat pancreatic acinar cells

In the cholecystokinin (CCK)hyperstimulation model of acute pancreatitis, two early intracellularevents, activation of trypsinogen and activation of nuclear factor-B(NF-B), are thought to be important in the development of thedisease. In this study, the relationship between these two events wasinvestigated. NF-B activity was monitored by using a DNA bindingassay and mob-1 chemokine gene expression. Intracellulartrypsin activity was measured by using a fluorogenic substrate.Protease inhibitors including FUT-175, Pefabloc, and E-64d preventedCCK stimulation of intracellular trypsinogen and NF-B activation.Likewise, the NF-B inhibitors pyrrolidine dithiocarbamate andN-acetyl-L-cysteine inhibited CCK stimulation ofNF-B and intracellular trypsinogen activation. These resultssuggested a possible codependency of these two events. However, CCKstimulated NF-B activation in Chinese hamster ovary-CCK_Acells, which do not express trypsinogen, indicating that trypsin is notnecessary for CCK activation of NF-B. Furthermore,adenovirus-mediated expression in acinar cells of active p65 subunitsto stimulate NF-B, or of inhibitory B- molecules to inhibitNF-B, did not affect either basal or CCK-mediated trypsinogenactivation. Thus trypsinogen and NF-B activation are independentevents stimulated by CCK.

     

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.

     

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.

     

Cholecystokinin induction of mob-1 chemokine expression in pancreatic acinar cells requires NF-kappa B activation

Inflammatory mediators are involved in the early phase of acutepancreatitis, but the cellular mechanisms responsible for theirgeneration within pancreatic cells are unknown. We examined the role ofnuclear factor-B (NF-B) in cholecystokinin octapeptide (CCK-8)-induced mob-1 chemokineexpression in pancreatic acinar cells in vitro. Supraphysiological, butnot physiological, concentrations of CCK-8 increased inhibitory B(IB-) degradation, NF-B activation, andmob-1 gene expression in isolatedpancreatic acinar cells. CCK-8-induced IB- degradation wasmaximal within 1 h. Expression ofmob-1 was maximal within 2 h. Neitherbombesin nor carbachol significantly increasedmob-1 mRNA or induced IB-degradation. Thus the concentration, time, and secretagogue dependenceof mob-1 gene expression and IB-degradation were similar. Inhibition of NF-B with pharmacologicalagents or by adenovirus-mediated expression of the inhibitory proteinIB- also inhibited mob-1 geneexpression. These data indicate that the NF-B signaling pathway isrequired for CCK-8-mediated induction ofmob-1 chemokine expression inpancreatic acinar cells. This supports the hypothesis that NF-Bsignaling is of central importance in the initiation of acute pancreatitis.

     

Expression of the TGF-beta receptor gene and sensitivity to growth inhibition following polyamine depletion

Our previous studieshave shown that inhibition of polyamine biosynthesis increases thesensitivity of intestinal epithelial cells to growth inhibition inducedby exogenous transforming growth factor- (TGF-). This study wentfurther to determine whether expression of the TGF- receptor genesis involved in this process. Studies were conducted in the IEC-6 cellline, derived from rat small intestinal crypt cells. Administration of-difluoromethylornithine (DFMO), a specific inhibitor of ornithinedecarboxylase (the rate-limiting enzyme for polyamine synthesis), for 4 and 6 days depleted cellular polyamines putrescine, spermidine, andspermine in IEC-6 cells. Polyamine depletion by DFMO increased levelsof the TGF- type I receptor (TGF-RI) mRNA and protein but had noeffect on the TGF- type II receptor expression. The inducedTGF-RI expression after polyamine depletion was associated with anincreased sensitivity to growth inhibition induced by exogenous TGF-but not by somatostatin. Extracellular matrix laminin inhibited IEC-6cell growth without affecting the TGF- receptor expression. Lamininconsistently failed to induce the sensitivity of TGF--mediatedgrowth inhibition. In addition, decreasing TGF-RI expression bytreatment with retinoic acid not only decreased TGF--mediated growthinhibition in normal cells but also prevented the increased sensitivityto exogenous TGF- in polyamine-deficient cells. These resultsindicate that 1) depletion of cellular polyamines by DFMOincreases expression of the TGF-RI gene and 2) increasedTGF-RI expression plays an important role in the process throughwhich polyamine depletion sensitizes intestinal epithelial cells togrowth inhibition induced by TGF-.

     

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.     

Serine phosphorylation of p60 tumor necrosis factor receptor by PKC-delta in TNF-alpha-activated neutrophils

Tumor necrosis factor-(TNF-) triggers degranulation and oxygen radical release in adherentneutrophils. The p60TNF receptor (p60TNFR) is responsible forproinflammatory signaling, and protein kinase C (PKC) is a candidatefor the regulation of p60TNFR. Both TNF- and the PKC-activatorphorbol 12-myristate 13-acetate triggered phosphorylation of p60TNFR.Receptor phosphorylation was on both serine and threonine but not ontyrosine residues. The PKC- isotype is a candidate enzyme for serinephosphorylation of p60TNFR. Staurosporine and the PKC- inhibitorrottlerin inhibited TNF--triggered serine but not threoninephosphorylation. Serine phosphorylation was associated withreceptor desensitization, as inhibition of PKC resulted in enhanceddegranulation (elastase release). After neutrophil activation, PKC-was the only PKC isotype that associated with p60TNFR within thecorrect time frame for receptor phosphorylation. In vitro, onlyPKC-, but not the -, I-, II-, or -isotypes, wascompetent to phosphorylate the receptor, indicating that p60TNFR is adirect substrate for PKC-. These findings suggest a selective rolefor PKC- in negative regulation of the p60TNFR and ofTNF--induced signaling.

     

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.

     

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.

     

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.

     

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.

     

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.

     

Differential translocation of protein kinase C isozymes by phorbol esters, EGF, and ANG II in rat liver WB cells

The protein kinaseC (PKC) family represents an important group of enzymes whoseactivation is associated with their translocation from the cytosol todifferent cellular membranes. In this study, the spatial distributionof PKC-, - and - in rat liver epithelial (WB) cells has beenexamined by Western blot analysis after subcellular fractionation.Cytosolic, membrane, nuclear, and cytoskeletal fractions were obtainedfrom cells stimulated with phorbol 12-myristate 13-acetate (PMA),angiotensin II (ANG II), or epidermal growth factor (EGF). PMA causedmost of the PKC-, - and - initially present in the cytosol tobe transported to the membrane and nuclear fractions. In contrast, bothANG II and EGF induced only a minor translocation of PKC- to themembrane fraction but caused a statistically significantmembrane-directed movement of PKC- and -. Translocation ofPKC- and - to the nucleus induced by ANG II and EGF was transient and quantitatively smaller than that induced by PMA. PKC- and -were present in the cytoskeleton of resting cells, but although PMA,ANG II, and EGF caused some changes in their content, these werevariable, suggesting that the cytoskeleton fraction was heterogeneous. PKC depletion inhibited ANG II-induced mitogenesis and the sustained activation of Raf-1 and extracellular regulated protein kinase (ERK).However, although PKC depletion inhibited EGF-induced mitogenesis, themaximum EGF-induced activation of the ERK pathway was only slightlyretarded. We hypothesize that PKC- and - are involved inmitogenesis via both ERK-dependent and ERK-independent mechanisms. These results support the notion that specific PKC isozymes exert spatially defined effects by virtue of their directed translocation todistinct intracellular sites.

     

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.     

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

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