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.

     

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.

     

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.

     

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.

     

Elevation of resting mitochondrial membrane potential of neural cells by cyclosporin A, BAPTA-AM, and bcl-2

This study testedthe hypothesis that the activity of the mitochondrial membranepermeability transition pore (PTP) affects the resting mitochondrialmembrane potential () of normal, healthy cells and that theanti-apoptotic gene product Bcl-2 inhibits the basal activity of thePTP. was measured by both fluorometric and nonfluorometricmethods with SY5Y human neuroblastoma cells and with GT1-7hypothalamic cells and PC12 pheochromocytoma cells in the absence andpresence of Bcl-2 gene overexpression. The resting of Bcl-2nonexpressing PC12 and wild-type SY5Y cells was increased significantlyby the presence of the PTP inhibitor cyclosporin A (CsA) or byintracellular Ca²⁺ chelation through exposure to theacetoxymethyl ester of1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid(BAPTA-AM). The of Bcl-2-overexpressing PC12 cells was largerthan that of Bcl-2-negative cells and not significantly increased byCsA or by Ca²⁺ chelation. CsA did not present a significanteffect on the monitored in unstressed GT1-7 cells but didinhibit the decrease in elicited by the addition oft-butyl hydroperoxide, an oxidative inducer of themitochondrial permeability transition. These results support thehypothesis that an endogenous PTP activity can contribute to loweringthe basal of some cells and that Bcl-2 can regulate theendogenous activity of the mitochondrial PTP.

     

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.     

Antisense oligonucleotide to PKC-epsilon alters cAMP-dependent stimulation of CFTR in Calu-3 cells

Protein kinase C(PKC) regulates cystic fibrosis transmembrane conductance regulator(CFTR) channel activity but the PKC signaling mechanism is not yetknown. The goal of these studies was to identify PKC isotype(s)required for control of CFTR function. CFTR activity was measured as³⁶Cl efflux in a Chinese hamsterovary cell line stably expressing wild-type CFTR (CHO-wtCFTR) and in aCalu-3 cell line. Chelerythrine, a PKC inhibitor, delayed increasedCFTR activity induced with phorbol 12-myristate 13-acetate or with thecAMP-generating agents ()-epinephrine or forskolin plus8-(4-chlorophenylthio)adenosine 3',5'- cyclicmonophosphate. Immunoblot analysis of Calu-3 cells revealed thatPKC-, -_II, -, -, and- were expressed in confluent cell cultures. Pretreatment of cellmonolayers with Lipofectin plus antisense oligonucleotide to PKC-for 48 h prevented stimulation of CFTR with ()-epinephrine,reduced PKC- activity in unstimulated cells by 52.1%, and decreasedPKC- mass by 76.1% but did not affect hormone-activated proteinkinase A activity. Sense oligonucleotide to PKC- and antisenseoligonucleotide to PKC- and - did not alter()-epinephrine-stimulated CFTR activity. These results demonstrate the selective regulation of CFTR function by constitutively active PKC-.

     

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-.

     

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.

     

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.

     

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.

     

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.

     

Actin filament organization is required for proper cAMP-dependent activation of CFTR

Previous studieshave indicated a role of the actin cytoskeleton in the regulation ofthe cystic fibrosis transmembrane conductance regulator (CFTR) ionchannel. However, the exact molecular nature of this regulation isstill largely unknown. In this report human epithelial CFTR wasexpressed in human melanoma cells genetically devoid of the filaminhomologue actin-cross-linking protein ABP-280 [ABP()]. cAMP stimulation of ABP() cells orcells genetically rescued with ABP-280 cDNA [ABP(+)] waswithout effect on whole cell Cl currents. InABP() cells expressing CFTR, cAMP was also without effect onCl conductance. In contrast, cAMP induced a 10-foldincrease in the diphenylamine-2-carboxylate (DPC)-sensitive whole cellCl currents of ABP(+)/CFTR(+) cells. Further, incells expressing both CFTR and a truncated form of ABP-280 unable tocross-link actin filaments, cAMP was also without effect on CFTRactivation. Dialysis of ABP-280 or filamin through the patch pipette,however, resulted in a DPC-inhibitable increase in the whole cellcurrents of ABP()/CFTR(+) cells. At the single-channel level,protein kinase A plus ATP activated single Clchannels only in excised patches from ABP(+)/CFTR(+) cells.Furthermore, filamin alone also induced Cl channelactivity in excised patches of ABP()/CFTR(+) cells. The presentdata indicate that an organized actin cytoskeleton is required forcAMP-dependent activation of CFTR.

     

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.

     

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.     

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.

     

TNF-alpha and insulin, alone and synergistically, induce plasminogen activator inhibitor-1 expression in adipocytes

Obesity is associated with hyperinsulinemia and elevatedconcentrations of tumor necrosis factor- (TNF-) inadipose tissue. TNF- has been implicated as an inducer of thesynthesis of plasminogen activator inhibitor-1 (PAI-1), the primaryphysiological inhibitor of fibrinolysis, mediated by plasminogenactivators in cultured adipocytes. To identify mechanism(s) throughwhich TNF- induces PAI-1, 3T3-L1 preadipocytes were differentiatedinto adipocytes and exposed to TNF- for 24 h. TNF- selectivelyincreased the synthesis of PAI-1 without increasing activity ofplasminogen activators. Both superoxide (generated by xanthine oxidaseplus hypoxanthine) and hydrogen peroxide were potent inducers of PAI-1, and hydroxyl radical scavengers completely abolished the TNF- induction of PAI-1. Exposure of adipocytes to TNF- or insulin aloneover 5 days increased PAI-1 production. These agonists exert synergistic effects. Results obtained suggest that TNF- stimulates PAI-1 production by adipocytes, an effect potentiated by insulin, andthat adipocyte generation of reactive oxygen centered radicals mediatesthe induction of PAI-1 production by TNF-. Because induction ofPAI-1 by TNF- is potentiated synergistically by insulin, both agonists appear likely to contribute to the impairment of fibrinolytic system capacity typical in obese, hyperinsulinemic patients.

     

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.

     

Exocytosis is not involved in activation of Cl- secretion via CFTR in Calu-3 airway epithelial cells

Cystic fibrosis iscaused by mutations in the cystic fibrosis transmembrane conductanceregulator (CFTR) Clchannel, which mediates transepithelialCl transport in a varietyof epithelia, including airway, intestine, pancreas, and sweat duct. Insome but not all epithelial cells, cAMP stimulatesCl secretion in part byincreasing the number of CFTRCl channels in the apicalplasma membrane. Because the mechanism whereby cAMP stimulates CFTRCl secretion is cell-typespecific, our goal was to determine whether cAMP elevates CFTR-mediatedCl secretion across serousairway epithelial cells by stimulating the insertion of CFTRCl channels from anintracellular pool into the apical plasma membrane. To this end westudied Calu-3 cells, a human airway cell line with a serous cellphenotype. Serous cells in human airways, such as Calu-3 cells, expresshigh levels of CFTR, secrete antibiotic-rich fluid, and play a criticalrole in airway function. Moreover, dysregulation of CFTR-mediatedCl secretion in serouscells is thought to contribute to the pathophysiology of cysticfibrosis lung disease. We report that cAMP activation of CFTR-mediatedCl secretion across humanserous cells involves stimulation of CFTR channels present in theapical plasma membrane and does not involve the recruitment of CFTRfrom an intracellular pool to the apical plasma membrane.

     

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.