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1.
Heterologous desensitization of response mediated by selective PKC-dependent phosphorylation of G(i-1) and G(i-2) 总被引:1,自引:0,他引:1
Murthy KS Grider JR Makhlouf GM 《American journal of physiology. Cell physiology》2000,279(4):C925-C934
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 Gi-1 and Gi-2,but not Gi-3 or Go, and blockedinhibition of adenylyl cyclase mediated by somatostatin receptorscoupled to Gi-1 and opioid receptors coupled toGi-2, but not by muscarinic M2 and adenosineA1 receptors coupled to Gi-3. Phosphorylationof Gi-1 and Gi-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 Gi-1 andGi-2, inhibited the PLC- response to somatostatin and[D-Pen2,5]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 ofGi-1 and Gi-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. 相似文献
2.
Carbachol-induced desensitization of PLC-beta pathway in rat myometrium: downregulation of Gqalpha /G11alpha 总被引:2,自引:0,他引:2
Lajat Sandrine; Harbon Simone; Tanfin Zahra 《American journal of physiology. Cell physiology》1998,275(3):C636
In the estrogen-treated rat myometrium, carbachol increased thegeneration of inositol phosphates by stimulating the muscarinic receptor-Gq/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 ofGq/G11.Atropine failed to induce desensitization as well asGq/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 ofGq/G11. Data suggest that a decrease in the level ofGq/G11is subsequent to its activation and may account forheterologous desensitization. 相似文献
3.
Interaction of alpha- and beta-subunits in native H-K-ATPase and cultured cells transfected with H-K-ATPase beta-subunit 总被引:1,自引:0,他引:1
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. 相似文献
4.
Qin Ning; Olcese Riccardo; Stefani Enrico; Birnbaumer Lutz 《American journal of physiology. Cell physiology》1998,274(5):C1324
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 neuronal1E voltage-gated calciumchannel expressed in Xenopus oocyteswas investigated without and with simultaneous coexpression of eitherthe 1b- or the2a-subunit. Most aspects of1E 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) for1E1b-channels. These results indicate that2 facilitates the expressionand the maturation of1E-channels and converts thesechannels into molecules responding more rapidly to voltage. 相似文献
5.
Role of beta 1- and beta 3-adrenoceptors in the regulation of lipolysis and thermogenesis in rat brown adipocytes 总被引:1,自引:0,他引:1
Atgie Claude; D'Allaire Francois; Bukowiecki Ludwik J. 《American journal of physiology. Cell physiology》1997,273(4):C1136
To evaluate the physiological functions of1-,2-, and3-adrenoceptors (ARs) in brownadipose tissue, the lipolytic and respiratory effects of variousadrenergic agonists and antagonists were studied in rat brownadipocytes. The -agonists stimulated both lipolysis and respiration(8-10 times above basal levels), with the following order ofpotency (concentration eliciting 50% of maximum response):CL-316243 (3) > BRL-37344(3) > isoproterenol (mainly1/2) > norepinephrine (NE; mainly1/2) > epinephrine (mainly1/2) dobutamine (1) procaterol (2). Schild plot coefficients of competitive inhibition experiments using ICI-89406 (1 antagonist) revealed thatmore than one type of receptor mediates NE action. It is concluded fromour results that 1) NE, at low plasma levels (1-25 nM), stimulates lipolysis and respiration mainly through 1-ARs,2) NE, at higher levels, stimulateslipolysis and respiration via both1- and3-ARs,3)2-ARs play only a minor role,and 4)3-ARs may represent thephysiological receptors for the high NE concentrations in the synapticcleft, where the high-affinity1-ARs are presumablydesensitized. It is also suggested that lipolysis represents theflux-generating step regulating mitochondrial respiration. 相似文献
6.
Bitter taste transduced by PLC-beta(2)-dependent rise in IP(3) and alpha-gustducin-dependent fall in cyclic nucleotides 总被引:5,自引:0,他引:5
Yan W Sunavala G Rosenzweig S Dasso M Brand JG Spielman AI 《American journal of physiology. Cell physiology》2001,280(4):C742-C751
Current evidence points to the existence of multiple processesfor bitter taste transduction. Previous work demonstrated involvement of the polyphosphoinositide system and an -gustducin(Ggust)-mediated stimulation of phosphodiesterase inbitter taste transduction. Additionally, a taste-enriched G protein-subunit, G13, colocalizes with Ggustand mediates the denatonium-stimulated production of inositol1,4,5-trisphosphate (IP3). Using quench-flow techniques, weshow here that the bitter stimuli, denatonium and strychnine, inducerapid (50-100 ms) and transient reductions in cAMP and cGMP andincreases in IP3 in murine taste tissue. This decrease ofcyclic nucleotides is inhibited by Ggust antibodies,whereas the increase in IP3 is not affected by antibodiesto Ggust. IP3 production is inhibited byantibodies specific to phospholipase C-2(PLC-2), a PLC isoform known to be activated byG-subunits. Antibodies to PLC-3 or toPLC-4 were without effect. These data suggest atransduction mechanism for bitter taste involving the rapid andtransient metabolism of dual second messenger systems, both mediatedthrough a taste cell G protein, likely composed ofGgust//13, with both systems beingsimultaneously activated in the same bitter-sensitive taste receptor cell. 相似文献
7.
Sun Lihyun; Weaver Debora J.; Amsler Kurt; Weiss Ellen R. 《American journal of physiology. Cell physiology》1998,274(4):C1030
LLC-PK1, an epithelial cellline derived from the kidney proximal tubule, was used to study theability of the G protein -subunit, Gq, to regulate celldifferentiation. A constitutively active mutant protein,qQ209L, was expressed using theLacSwitch-inducible mammalian expression system. Induction ofqQ209L 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 ofqQ209L 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 inqQ209L-expressing cells,suggesting that the regulation of PKC byGq 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 inqQ209L-expressing cells. These data implicate PKC and PKC in the pathway used byGq to block the development ofNa+-dependent hexose transport inIPTG-treated cells. 相似文献
8.
Chalfant Michael L.; Denton Jerod S.; Berdiev Bakhram K.; Ismailov Iskander I.; Benos Dale J.; Stanton Bruce A. 《American journal of physiology. Cell physiology》1999,276(2):C477
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(pHi) but not by changes inextracellular pH (pHo).Acidification reduced and alkalization increased channel activity by avoltage-independent mechanism. Moreover, a reduction ofpHi 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 thatpHi but notpHo regulates ENaC and that the-ENaC subunit is regulated directly bypHi. 相似文献
9.
Sangan Pitchai; Kolla Sarah S.; Rajendran Vazhaikkurichi M.; Kashgarian Michael; Binder Henry J. 《American journal of physiology. Cell physiology》1999,276(2):C350
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. 相似文献
10.
Ji Hong-Long; Fuller Catherine M.; Benos Dale J. 《American journal of physiology. Cell physiology》1998,275(5):C1182
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(Ki; 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. 相似文献
11.
Tumor necrosis factor- (TNF-), oneof the major inflammatory cytokines, is known to influence endothelialcell migration. In this study, we demonstrate that exposure of calfpulmonary artery endothelial cells to TNF- caused an increase in theformation of membrane protrusions and cell migration. Fluorescencemicroscopy revealed an increase in v3focal contacts but a decrease in 51 focalcontacts in TNF--treated cells. In addition, both cell-surface andtotal cellular expression of v3-integrinsincreased significantly, whereas the expression of51-integrins was unaltered. Only focalcontacts containing v3- but not51-integrins were present in membraneprotrusions of cells at the migration front. In contrast, robust focalcontacts containing 51-integrins were present in cells behind the migration front. A blocking antibody tov3, but not a blocking antibody to5-integrins, significantly inhibited TNF--inducedcell migration. These results indicate that in response to TNF-,endothelial cells may increase the activation and ligation ofv3 while decreasing the activation andligation of 51-integrins to facilitatecell migration, a process essential for vascular wound healing and angiogenesis. 相似文献
12.
Protein kinase Calpha participates in activation of store-operated Ca2+ channels in human glomerular mesangial cells 总被引:2,自引:0,他引:2
Protein kinase C (PKC) plays animportant role in activating store-operated Ca2+ channels(SOC) in human mesangial cells (MC). The present study was performed todetermine the specific isoform(s) of conventional PKC involved inactivating SOC in MC. Fura 2 fluorescence ratiometry showed that thethapsigargin-induced Ca2+ entry (equivalent to SOC) wassignificantly inhibited by 1 µM Gö-6976 (a specific PKC andI inhibitor) and PKC antisense treatment (2.5 nM for 24-48h). However, LY-379196 (PKC inhibitor) and2,2',3,3',4,4'-hexahydroxy-1,1'-biphenyl-6,6'-dimethanoldimethyl ether(HBDDE; PKC and inhibitor) failed to affect thapsigargin-evoked activation of SOC. Single-channel analysis in the cell-attached configuration revealed that Gö-6976 and PKC antisensesignificantly depressed thapsigargin-induced activation of SOC.However, LY-379196 and HBDDE did not affect the SOC responses. Ininside-out patches, application of purified PKC or I, but notII or , significantly rescued SOC from postexcision rundown.Western blot analysis revealed that thapsigargin evoked a decrease incytosolic expression with a corresponding increase in membraneexpression of PKC and . However, the translocation from cytosolto membranes was not detected for PKCI or II. These resultssuggest that PKC participates in the intracellular signaling pathwayfor activating SOC upon release of intracellular stores ofCa2+. 相似文献
13.
Morris David J.; Latif Syed A.; Rokaw Michael D.; Watlington Charles O.; Johnson John P. 《American journal of physiology. Cell physiology》1998,274(5):C1245
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(Isc)] inA6 cells. All of these 11-HSD inhibitory substances induced asignificant increment in corticosterone-inducedIsc, which wasdetectable within 2 h. However, none of these agents caused an increasein Isc whenincubated by themselves with A6 cells. In all cases, the additionalIsc was inhibitedby the mineralocorticoid receptor (MR) antagonist, RU-28318, whereasthe original Iscelicited 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 Iscin the one inhibitor tested. Troleandomycin, a selective inhibitor ofCYP3A, inhibited 6-hydroxylase and also significantly enhancedcorticosterone-induced Isc at 2 h. Theseexperiments indicate that the enhanced MR-mediated Isc 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). 相似文献
14.
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-. 相似文献
15.
Upregulation of alpha(8)beta(1)-integrin in cardiac fibroblast by angiotensin II and transforming growth factor-beta1 总被引:3,自引:0,他引:3
Thibault G Lacombe MJ Schnapp LM Lacasse A Bouzeghrane F Lapalme G 《American journal of physiology. Cell physiology》2001,281(5):C1457-C1467
Using a novel pharmacological tool with125I-echistatin to detect integrins on the cell, we haveobserved that cardiac fibroblasts harbor five different RGD-bindingintegrins: 81,31, 51, v1, and v3.Stimulation of cardiac fibroblasts by angiotensin II (ANG II) ortransforming growth factor-1 (TGF-1) resulted in an increase ofprotein and heightening by 50% of the receptor density of81-integrin. The effect of ANG II wasblocked by an AT1, but not an AT2, receptorantagonist, or by an anti-TGF-1 antibody. ANG II and TGF-1increased fibronectin secretion, smooth muscle -actin synthesis, andformation of actin stress fibers and enhanced attachment of fibroblaststo a fibronectin matrix. The 8- and1-subunits were colocalized by immunocytochemistry with vinculin or 3-integrin at focal adhesion sites.These results indicate that 81-integrinis an abundant integrin on rat cardiac fibroblasts. Its positivemodulation by ANG II and TGF-1 in a myofibroblast-likephenotype suggests the involvement of81-integrin in extracellularmatrix protein deposition and cardiac fibroblast adhesion. 相似文献
16.
beta-adrenergic receptor/cAMP-mediated signaling and apoptosis of S49 lymphoma cells 总被引:6,自引:0,他引:6
Yan L Herrmann V Hofer JK Insel PA 《American journal of physiology. Cell physiology》2000,279(5):C1665-C1674
-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 Gs and functional protein kinase A(PKA), respectively, AR activation of Gs 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 Gs 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. 相似文献
17.
beta-amyloid-induced migration of monocytes across human brain endothelial cells involves RAGE and PECAM-1 总被引:8,自引:0,他引:8
Giri R Shen Y Stins M Du Yan S Schmidt AM Stern D Kim KS Zlokovic B Kalra VK 《American journal of physiology. Cell physiology》2000,279(6):C1772-C1781
In patients withamyloid -related cerebrovascular disorders, e.g., Alzheimer'sdisease, one finds increased deposition of amyloid peptide (A) andincreased presence of monocyte/microglia cells in the brain. However,relatively little is known of the role of A in the trafficking ofmonocytes across the blood-brain barrier (BBB). Our studies show thatinteraction of A1-40 with monolayer of human brainendothelial cells results in augmented adhesion and transendothelialmigration of monocytic cells (THP-1 and HL-60) and peripheral bloodmonocytes. The A-mediated migration of monocytes was inhibited byantibody to A receptor (RAGE) and platelet endothelial cell adhesionmolecule (PECAM-1). Additionally, A-induced transendothelialmigration of monocytes were inhibited by protein kinase C inhibitor andaugmented by phosphatase inhibitor. We conclude that interaction ofA with RAGE expressed on brain endothelial cells initiates cellularsignaling leading to the transendothelial migration of monocytes. Wesuggest that increased diapedesis of monocytes across the BBB inresponse to A present either in the peripheral circulation or in thebrain parenchyma may play a role in the pathophysiology of A-relatedvascular disorder. 相似文献
18.
Sangan P Thevananther S Sangan S Rajendran VM Binder HJ 《American journal of physiology. Cell physiology》2000,278(1):C182-C189
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 aKm 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. 相似文献
19.
Clathrin and the -adaptin subunit of the AP-1 clathrinadaptor have been previously identified on H-K-ATPase-richtubulovesicles from gastric acid secretory (oxyntic) cells [C. T. Okamoto, S. M. Karam, Y. Y. Jeng, J. G. Forte, and J. Goldenring.Am. J. Physiol. 274 (Cell Physiol. 43):C1017-C1029]. We further characterized this AP-1 adaptorfrom rabbit and hog tubulovesicles biochemically and immunologically.Clathrin coat proteins were stripped from purified tubulovesicularmembranes and fractionated by hydroxyapatite chromatography. The AP-1adaptor appears to elute at 200 mM sodium phosphate, based on thepresence of proteins in this fraction that are immunoreactive withantibodies against three of the four subunits of this heterotetramericcomplex: the -, µ1-, and1-adaptin subunits. Althoughthe putative -adaptin subunit in this fraction is not immunoreactivewith the anti--adaptin monoclonal antibody (MAb), this -adaptinis immunoreactive with polyclonal antibodies (PAbs) directed againstthe peptide sequenceGly625-Asp-Leu-Leu-Gly-Asp-Leu-Leu-Asn-Leu-Asp-Leu-Gly-Pro-Pro-Val640,a region conserved between 1-and 2-adaptins that is thought to be involved in the binding of clathrin heavy chain.Immunoprecipitation of the AP-1 adaptor complex from this fraction withanti--adaptin MAb 100/3 resulted in the coimmunoprecipitation of the-adaptin that did not react with the anti--adaptin MAb but didreact with the anti--adaptin PAbs. In contrast, immunoprecipitationof the AP-1 adaptor complex from crude clathrin-coated vesicles from brain resulted in the coimmunoprecipitation of a -adaptin that wasrecognized by both the anti--adaptin MAb and PAbs. These resultssuggest that the tubulovesicular AP-1 adaptor complex may be distinctfrom that found in the trans-Golgi network and may contain animmunologically distinct -adaptin. This immunologically distinct-adaptin may be diagnostic of apical tubulovesicular endosomes ofepithelial cells. 相似文献
20.
Awayda Mouhamed S.; Tousson Albert; Benos Dale J. 《American journal of physiology. Cell physiology》1997,273(6):C1889
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 (Po) 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 ofPo. 相似文献