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-.

     

Antisense oligodeoxynucleotide to PKC-delta blocks alpha 1-adrenergic activation of Na-K-2Cl cotransport

A role for protein kinase C (PKC)- and -isotypes in ₁-adrenergicregulation of human tracheal epithelial Na-K-2Cl cotransport wasstudied with the use of isotype-specific PKC inhibitors and antisenseoligodeoxynucleotides to PKC- or - mRNA. Rottlerin, a PKC-inhibitor, blocked 72% of basolateral-to-apical, bumetanide-sensitive ³⁶Cl flux innystatin-permeabilized cell monolayers stimulated with methoxamine, an₁-adrenergic agonist, with a50% inhibitory concentration of 2.3 µM. Methoxamine increased PKCactivity in cytosol and a particulate fraction; the response wasinsensitive to PKC- and -_IIisotype-specific inhibitors, but was blocked by general PKC inhibitorsand rottlerin. Rottlerin also inhibited methoxamine-induced PKCactivity in immune complexes of PKC-, but not PKC-. At the subcellular level, methoxamine selectively elevated cytosolic PKC-activity and particulate PKC- activity. Pretreatment of cellmonolayers with antisense oligodeoxynucleotide to PKC- for 48 hreduced the amount of whole cell and cytosolic PKC-, diminished whole cell and cytosolic PKC- activity, and blockedmethoxamine-stimulated Na-K-2Cl cotransport. Sense oligodeoxynucleotideto PKC- and antisense oligodeoxynucleotide to PKC- did not altermethoxamine-induced cotransport activity. These results demonstrate theselective activation of Na-K-2Cl cotransport by cytosolic PKC-.

     

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.

     

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

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

     

Inhibitory actions of ceramide upon PKC-epsilon/ERK interactions

We have previously shown that interleukin-1receptor-generated ceramide induces growth arrest in smooth musclepericytes by inhibiting an upstream kinase in the extracellularsignal-regulated kinase (ERK) cascade. Here, we now report themechanism by which ceramide inhibits ERK activity. Ceramide renders thehuman embryonic kidney 293 cells (HEK 293) resistant to the mitogenicactions of growth factors and activators of protein kinase C (PKC). A role for PKC to mediate ceramide inhibition of growth factor-induced ERK activity and mitogenesis is suggested, as exogenous ceramide directly inhibits both immunoprecipitated and recombinant PKC- activities. To confirm that PKC- is necessary for ceramide-inhibited ERK activity, HEK 293 cells were transfected with a dominant-negative mutant of PKC- (PKC-). These transfected cells respond toinsulin-like growth factor I (IGF-I) with a significantly decreased ERKactivity that is not further reduced by ceramide treatment.Coimmunoprecipitation studies reveal that the treatment with IGF-Iinduces the association of ERK with PKC- but not with PKC-.Ceramide treatment significantly inhibits the IGF-I-induced PKC-interaction with bioactive phosphorylated ERK. Ceramide also inhibitsIGF-I-induced PKC- association with Raf-1, an upstream kinase ofERK. Together, these studies demonstrate that ceramide exertsanti-mitogenic actions by limiting the ability of PKC- to form asignaling complex with Raf-1 and ERK.

     

Dopamine-induced translocation of protein kinase C isoforms visualized in renal epithelial cells

Short-term regulation of sodiummetabolism is dependent on the modulation of the activity of sodiumtransporters by first and second messengers. In understanding diseasesassociated with sodium retention, it is necessary to identify thecoupling between these messengers. We have examined whether dopamine,an important first messenger in tubular cells, activates andtranslocates various protein kinase C (PKC) isoforms. We used aproximal tubular-like cell line, LLCPK-1 cells, in which dopamine wasfound to inhibit Na⁺-K⁺-ATPase in aPKC-dependent manner. Translocation of PKC isoforms was studied withboth subcellular fractionation and confocal microscopy. Both techniquesrevealed a dopamine-induced translocation from cytosol to plasmamembrane of PKC- and -, but not of PKC-, -, and -. Theprocess of subcellular fractionation resulted in partial translocationof PKC-. This artifact was eliminated in confocal studies. Confocalimaging permitted detection of translocation within 20 s.Translocation was abolished by a phospholipase C inhibitor and by anantagonist against the dopamine 1 subtype (D₁) but not the2 subtype of receptor (D₂). In conclusion, this studyvisualizes in renal epithelial cells a very rapid activation of thePKC- and - isoforms by the D₁ receptor subtype.

     

Stability of actin cytoskeleton and PKC-delta binding to actin regulate NKCC1 function in airway epithelial cells

Activation of airwayepithelial Na-K-2Cl cotransporter (NKCC)1 requires increased activityof protein kinase C (PKC)-, which localizes predominantly to theactin cytoskeleton. Prompted by reports of a role for actin in NKCC1function, we studied a signaling mechanism linking NKCC1 and PKC.Stabilization of actin polymerization with jasplakinolide increasedactivity of NKCC1, whereas inhibition of actin polymerization withlatrunculin B prevented hormonal activation of NKCC1. Protein-proteininteractions among NKCC1, actin, and PKC- were verified by Westernblot analysis of immunoprecipitated proteins. PKC- was detected inimmunoprecipitates of NKCC1 and vice versa. Actin was also detected inimmunoprecipitates of NKCC1 and PKC-. Pulldown of endogenous actinrevealed the presence of NKCC1 and PKC-. Binding of recombinantPKC- to NKCC1 was not detected in overlay assays. Rather, activatedPKC- bound to actin, and this interaction was prevented by a peptideencoding C2, a C2-like domain based on the amino acid sequence ofPKC-. C2 also blocked stimulation of NKCC1 function bymethoxamine. Immunofluorescence and confocal microscopy revealedPKC- in the cytosol and cell periphery. Merged images of cellsstained for actin and PKC- indicated colocalization of PKC- andactin at the cell periphery. The results indicate that actin iscritical for the activation of NKCC1 through a direct interaction with PKC-.

     

Acute glucose-induced downregulation of PKC-betaII accelerates cultured VSMC proliferation

Accelerated vascular smooth muscle cell(VSMC) proliferation contributes to the formation of atheroscleroticlesions. To investigate protein kinase C (PKC)-II functions withregard to glucose-induced VSMC proliferation, human VSMC from aorta(AoSMC), a clonal VSMC line of rat aorta (A10), and A10 cellsoverexpressing PKC-I (I-A10) and PKC-II (II-A10) werestudied with the use of three techniques to evaluate glucose effects onaspects affecting proliferation. High glucose (25 mM) increased DNAsynthesis and accelerated cell proliferation compared with normalglucose (5.5 mM) in AoSMC and A10 cells, but not in I-A10 andII-A10 cells. The PKC-II specific inhibitor CGP-53353 inhibitedglucose-induced cell proliferation and DNA synthesis in AoSMC and A10cells. In flow cytometry analysis, high glucose increased thepercentage of A10 cells at 12 h after cell cycle initiation butdid not increase the percentage of I-A10 or II-A10 cells enteringS phase. PKC-II protein levels decreased before the peak of DNAsynthesis, and high glucose further decreased PKC-II mRNA andprotein levels in AoSMC and A10 cells. These results suggest that highglucose downregulates endogenous PKC-II, which then alters thenormal inhibitory role of PKC-II in cell cycle progression,resulting in the stimulation of VSMC proliferation through acceleration of the cell cycle.

     

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.

     

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.

     

Evidence for functional role of epsilonPKC isozyme in the regulation of cardiac Na(+) channels

Investigation of the role ofindividual protein kinase C (PKC) isozymes in the regulation ofNa⁺ channels has been largely limited by the lack ofisozyme-selective modulators. Here we used a novel peptide-specificactivator (V1-7) of PKC and other peptide isozyme-specificinhibitors in addition to the general PKC activator phorbol12-myristate 13-acetate (PMA) to dissect the role of individual PKCs inthe regulation of the human cardiac Na⁺ channel hH1,heterologously expressed in Xenopus oocytes. Peptides wereinjected individually or in combination into the oocyte. Whole cellNa⁺ current (I_Na) was recorded usingtwo-electrode voltage clamp. V1-7 (100 nM) and PMA (100 nM)inhibited I_Na by 31 ± 5% and 44 ± 8% (at 20 mV), respectively. These effects were not seen with thescrambled peptide for V1-7 (100 nM) or the PMA analog4-phorbol 12,13-didecanoate (100 nM). However, V1-7-and PMA-induced I_Na inhibition was abolished byV1-2, a peptide-specific antagonist of PKC. Furthermore,PMA-induced I_Na inhibition was not altered by100 nM peptide-specific inhibitors for -, -, -, or PKC. PMAand V1-7 induced translocation of PKC from soluble toparticulate fraction in Xenopus oocytes. This translocationwas antagonized by V1-2. In native rat ventricular myocytes,PMA and V1-7 also inhibited I_Na; thisinhibition was antagonized by V1-2. In conclusion, the resultsprovide evidence for selective regulation of cardiac Na⁺channels by PKC isozyme.

     

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.

     

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.     

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.

     

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.

     

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.     

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.

     

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.

     

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.

     

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.