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1.
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
2.
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
3.
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
4.
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
5.
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
6.
Rat sublingual glandM1 and M3 muscarinic receptors each directlyactivate exocrine secretion. To investigate the functional role ofcoreceptor expression, we determined receptor-G protein coupling.Although membrane proteins of 40 and 41 kDa are ADP-ribosylated bypertussis toxin (PTX), and 44 kDa proteins by cholera toxin (CTX), bothcarbachol-stimulated high-affinity GTPase activity and the GTP-inducedshift in agonist binding are insensitive to CTX or PTX. Carbacholenhances photoaffinity labeling([-32P]GTP-azidoaniline) of only 42-kDa proteins thatare subsequently tractable to immunoprecipitation by antibodiesspecific for Gq or G11 but notG12 or G13. Carbachol-stimulatedphotoaffinity labeling as well as phosphatidylinositol 4,5-bisphosphate(PIP2) hydrolysis is reduced 55% and 60%, respectively,by M1 receptor blockade with m1-toxin.Gq/11-specific antibody blocks carbachol-stimulated PIP2 hydrolysis. We also provide estimates of the molarratios of receptors to Gq and G11.Although simultaneous activation of M1 and M3receptors is required for a maximal response, both receptor subtypesare coupled to Gq and G11 to stimulateexocrine secretion via redundant mechanisms. 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.
Di Fusco F Hashim S Anand-Srivastava MB 《American journal of physiology. Cell physiology》2000,279(4):C990-C998
We haverecently reported enhanced levels of Gi proteins ingenetic and other experimentally induced models of hypertension, whereas the levels of Gs were decreased in hypertensiverats expressing cardiac hypertrophy. The present studies wereundertaken to investigate whether the decreased levels ofGs are associated with cardiac hypertrophy per se andused an aortocaval fistula (AV shunt; volume overload) rat model thatexclusively expresses cardiac hypertrophy. Cardiac hypertrophy inSprague-Dawley rats (200-250 g) was induced under anesthesia, and,after a period of 10 days, the hearts were used for adenylyl cyclaseactivity determination, protein quantification, and mRNA leveldetermination. A temporal relationship between the expression ofGs proteins and cardiac hypertrophy was also examined ondays 2, 3, 7, and 10 after induction of AV shuntin the rat. The heart-to-body-weight ratio (mg/g) was significantlyincreased in AV shunt rats after 3, 7, and 10 days of induction of AVshunt compared with sham-operated controls, whereas arterial bloodpressure was not different between the two groups. Guanosine5'-O-(3-thiotriphosphate) (GTPS) stimulated adenylylcyclase activity in a concentration-dependent manner in heart membranesfrom both groups; however, the degree of stimulation was significantlydecreased in AV shunt rats. In addition, the stimulatory effects ofisoproterenol were also diminished in AV shunt rats compared withcontrol rats, whereas glucagon-stimulated adenylyl cyclase activity wasnot different in the two groups. The inhibitory effects of oxotremorine(receptor-dependent Gi functions) and low concentrations ofGTPS on forskolin-stimulated adenylyl cyclase activity(receptor-independent Gi functions) were not different inthe two groups. In addition forskolin and NaF also stimulated adenylylcyclase activity to a lesser degree in AV shunt rats compared withcontrol rats. The levels of Gi-2 and Gi-3proteins and mRNA, as determined by immunoblotting and Northernblotting, respectively, were not different in both groups; however, thelevels of Gs45 andGs47, and not ofGs52, proteins were significantly decreasedin AV shunt rats by days 7 and 10 compared withcontrol rats, whereas no change was observed on days 2 and3 after induction of AV shunt. These results suggest thatthe decreased expression of Gs proteins may not be thecause but the effect of hypertrophy and that the diminishedresponsiveness of adenylyl cyclase to GTPS, isoproterenol, NaF, andforskolin in hearts from AV shunt rats may partly be due to thedecreased expression of Gs. It can be concluded fromthese studies that the decreased expression of Gs may beassociated with cardiac hypertrophy and not with arterial hypertension. 相似文献
9.
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
10.
Kitamura Kenichiro; Shiraishi Naoki; Singer William D.; Handlogten Mary E.; Tomita Kimio; Miller R. Tyler 《American journal of physiology. Cell physiology》1999,276(4):C930
Endothelin (ET) receptors activate heterotrimeric G proteinsthat are members of the Gi,Gq, andGs families but may also activatemembers of other families such asG12/13.G13 has multiple complexcellular effects that are similar to those of ET. We studied theability of ET receptors to activateG13 using an assay for Gprotein -chain activation that is based on the fact that an activated (GTP-bound) -chain is resistant to trypsinization compared with an inactive (GDP-bound) -chain. Nonhydrolyzable guanine nucleotides and AlMgF protectedG13 from degradation bytrypsin. In membranes from human embryonic kidney 293 cells thatcoexpress ETB receptors and13, ET-3 and5'-guanylylimidodiphosphate [Gpp(NH)p] increased theprotection of 13 compared withGpp(NH)p alone. The specificity ofETBreceptor-13 coupling wasdocumented by showing that 2receptors and isoproterenol or ETAreceptors and ET-1 did not activate13 and that a specificantagonist for ETB receptorsblocked ET-3-dependent activation of13. 相似文献
11.
Yorek Mark A.; Dunlap Joyce A.; Thomas Michael J.; Cammarata Patrick R.; Zhou Cheng; Lowe William L. Jr. 《American journal of physiology. Cell physiology》1998,274(1):C58
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. 相似文献
12.
Bubien James K.; Cornwell Trudy; Bradford Anne Lynn; Fuller Catherine M.; DuVall Michael D.; Benos Dale J. 《American journal of physiology. Cell physiology》1998,275(3):C702
Two independentsignal transduction pathways regulate lymphocyte amiloride-sensitivesodium channels (ASSCs), one utilizing cAMP as a second messenger andthe other utilizing a GTP-binding protein. This implies that two plasmamembrane receptors play a role in the regulation of lymphocyte ASSCs.In this study, we tested the hypothesis that1- and2-adrenergic receptorsindependently regulate lymphocyte ASSCs via the two previouslyidentified second messengers. Direct measurements indicated thatnorepinephrine increased lymphocyte cAMP and activated ASSCs. The2-specific inhibitor,yohimbine, blocked this activation, thereby linking 2-adrenergic receptors to ASSCregulation via cAMP. The1-specific ligand, terazosin,acted as an agonist and activated lymphocyte ASSCs but inhibited ASSCcurrent that had been preactivated by norepinephrine or8-(4-chlorophenylthio) (CPT)-cAMP. Terazosin had no effect on thelymphocyte whole cell ASSC currents preactivated by treatment withpertussis toxin. This finding indirectly links 1-adrenergic receptors tolymphocyte ASSC regulation via GTP-binding proteins. Terazosin had nodirect inhibitory or stimulatory effects on ,,-endothelialsodium channels reconstituted into planar lipid bilayers and expressedin Xenopus oocytes, ruling out a direct interaction between terazosin and the channels. These findings support the hypothesis that both1- and2-adrenergic receptors independently regulate lymphocyte ASSCs via GTP-binding proteins andcAMP, respectively. 相似文献
13.
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
14.
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
15.
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
16.
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
17.
Chen Pei-Xian; Mathews Paul M.; Good Peter J.; Rossier Bernard C.; Geering Kathi 《American journal of physiology. Cell physiology》1998,275(1):C139
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. 相似文献
18.
Tewari KP Malinowska DH Sherry AM Cuppoletti J 《American journal of physiology. Cell physiology》2000,279(1):C40-C50
An HEK-293 cell line stably expressing the humanrecombinant ClC-2 Cl channel was used in patch-clampstudies to study its regulation. The relative permeabilityPx/PCl calculated fromreversal potentials was I > Cl = NO3 = SCNBr. Theabsolute permeability calculated from conductance ratios wasCl = Br = NO3 SCN > I. The channel was activatedby cAMP-dependent protein kinase (PKA), reduced extracellular pH, oleicacid (C:18 cis9), elaidic acid (C:18trans9), arachidonic acid (AA; C:20cis5,8,11,14), and by inhibitors of AA metabolism,5,8,11,14-eicosatetraynoic acid (ETYA; C:20trans5,8,11,14),-methyl-4-(2-methylpropyl)benzeneacetic acid (ibuprofen), and2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2Cl channels were activated by a combination of forskolinplus IBMX and were inhibited by the cell-permeant myristoylated PKAinhibitor (mPKI). Channel activation by reduction of bath pH wasincreased by PKA and prevented by mPKI. AA activation of the ClC-2Cl channel was not inhibited by mPKI or staurosporine andwas therefore independent of PKA or protein kinase C activation. 相似文献
19.
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
20.
Overexpression of protein kinase C-delta increases tight junction permeability in LLC-PK1 epithelia 总被引:2,自引:0,他引:2
Mullin James M.; Kampherstein Jennifer A.; Laughlin Kathleen V.; Clarkin Cheryl E.K.; Miller R. Daniel; Szallasi Zoltan; Kachar Bechara; Soler Alejandro Peralta; Rosson Dan 《American journal of physiology. Cell physiology》1998,275(2):C544
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(RT) in cellsheets overexpressing PKC- was only 20% of that in cell sheetsincubated in the presence of tetracycline, in which the amount ofPKC- and RTwere 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 theRT 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 reducedRT, 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. 相似文献