共查询到20条相似文献,搜索用时 31 毫秒
1.
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
2.
Golovina VA Song H James PF Lingrel JB Blaustein MP 《American journal of physiology. Cell physiology》2003,284(2):C475-C486
The role of the Na+ pump2-subunit in Ca2+ signaling was examined inprimary cultured astrocytes from wild-type(2+/+ = WT) mouse fetuses and thosewith a null mutation in one [2+/ = heterozygote (Het)] or both [2/ = knockout (KO)] 2 genes. Na+ pump catalytic() subunit expression was measured by immunoblot; cytosol[Na+] ([Na+]cyt) and[Ca2+] ([Ca2+]cyt) weremeasured with sodium-binding benzofuran isophthalate and fura 2 byusing digital imaging. Astrocytes express Na+ pumpswith both 1- (80% of total ) and2- (20% of total ) subunits. Het astrocytesexpress 50% of normal 2; those from KO express none.Expression of 1 is normal in both Het and KO cells.Resting [Na+]cyt = 6.5 mM in WT, 6.8 mMin Het (P > 0.05 vs. WT), and 8.0 mM in KO cells(P < 0.001); 500 nM ouabain (inhibits only2) equalized [Na+]cyt at 8 mMin all three cell types. Resting[Ca2+]cyt = 132 nM in WT, 162 nM in Het,and 196 nM in KO cells (both P < 0.001 vs. WT).Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER)Ca2+ pumps and unloads the ER, induces transient (inCa2+-free media) or sustained (in Ca2+-repletemedia) elevation of [Ca2+]cyt. TheseCa2+ responses to 10 µM CPA were augmented in Het as wellas KO cells. When CPA was applied in Ca2+-free media, thereintroduction of Ca2+ induced significantly largertransient rises in [Ca2+]cyt (due toCa2+ entry through store-operated channels) in Het and KOcells than in WT cells. These results correlate with published evidencethat 2 Na+ pumps andNa+/Ca2+ exchangers are confined to plasmamembrane microdomains that overlie the ER. The data suggest thatselective reduction of 2 Na+ pump activitycan elevate local [Na+] and, viaNa+/Ca2+ exchange, [Ca2+] in thetiny volume of cytosol between the plasma membrane and ER. This, inturn, augments adjacent ER Ca2+ stores and therebyamplifies Ca2+ signaling without elevating bulk[Na+]cyt. 相似文献
3.
Astrocytes from Na(+)-K(+)-Cl(-) cotransporter-null mice exhibit absence of swelling and decrease in EAA release 总被引:1,自引:0,他引:1
Su G Kintner DB Flagella M Shull GE Sun D 《American journal of physiology. Cell physiology》2002,282(5):C1147-C1160
We reported previously that inhibition ofNa+-K+-Cl cotransporter isoform 1 (NKCC1) by bumetanide abolishes high extracellular K+concentration ([K+]o)-induced swelling andintracellular Cl accumulation in rat cortical astrocytes.In this report, we extended our study by using cortical astrocytes fromNKCC1-deficient (NKCC1/) mice. NKCC1 protein andactivity were absent in NKCC1/ astrocytes.[K+]o of 75 mM increased NKCC1 activityapproximately fourfold in NKCC1+/+ cells (P < 0.05) but had no effect in NKCC1/ astrocytes.Intracellular Cl was increased by 70% inNKCC1+/+ astrocytes under 75 mM[K+]o (P < 0.05) butremained unchanged in NKCC1/ astrocytes. Baselineintracellular Na+ concentration([Na+]i) in NKCC1+/+ astrocyteswas 19.0 ± 0.5 mM, compared with 16.9 ± 0.3 mM[Na+]i in NKCC1/ astrocytes(P < 0.05). Relative cell volume ofNKCC1+/+ astrocytes increased by 13 ± 2% in 75 mM[K+]o, compared with a value of 1.0 ± 0.5% in NKCC1/ astrocytes (P < 0.05).Regulatory volume increase after hypertonic shrinkage was completelyimpaired in NKCC1/ astrocytes.High-[K+]o-induced 14C-labeledD-aspartate release was reduced by ~30% inNKCC1/ astrocytes. Our study suggests that stimulationof NKCC1 is required for high-[K+]o-inducedswelling, which contributes to glutamate release from astrocytes underhigh [K+]o. 相似文献
4.
Jayaraman Sujatha; Biwersi Joachim; Verkman A. S. 《American journal of physiology. Cell physiology》1999,276(3):C747
The fluorescence of quinolinium-basedCl indicators such as6-methoxy-N-(3-sulfopropyl)quinolinium(SPQ) is quenched by Cl bya collisional mechanism without change in spectral shape. A series of"chimeric" dual-wavelengthCl indicators weresynthesized by conjugatingCl-sensitive and-insensitive chromophores with spacers. The SPQ chromophore(N-substituted 6-methoxyquinolinium; MQ) was selected as theCl-sensitive moiety[excitation wavelength(ex) 350 nm, emission wavelength (em) 450 nm]. N-substituted 6-aminoquinolinium (AQ) waschosen as theCl-insensitive moietybecause of its different spectral characteristics (ex 380 nm,em 546 nm), insensitivity toCl, positive charge (tominimize quenching by chromophore stacking/electron transfer), andreducibility (for noninvasive cell loading). The dual-wavelengthindicators were stable and nontoxic in cells and were distributeduniformly in cytoplasm, with occasional staining of the nucleus. Thebrightest and mostCl-sensitive indicatorswere -MQ-'-dimethyl-AQ-xylene dichloride andtrans-1,2-bis(4-[1-'-MQ-1'-'-dimethyl-AQ-xylyl]-pyridinium)ethylene (bis-DMXPQ). At 365-nm excitation, emission maxima were at 450 nm(Cl sensitive; Stern-Volmerconstants 82 and 98 M1)and 565 nm (Clinsensitive). Cystic fibrosis transmembrane conductanceregulator-expressing Swiss 3T3 fibroblasts were labeled with bis-DMXPQby hypotonic shock or were labeled with its uncharged reduced form(octahydro-bis-DMXPQ) by brief incubation (20 µM, 10 min). Changes inCl concentration inresponse to Cl/nitrateexchange were recorded by emission ratio imaging (450/565 nm) at 365-nmexcitation wavelength. These results establish a first-generation setof chimeric bisquinoliniumCl indicators forratiometric measurement ofCl concentration. 相似文献
5.
CFTR induces the expression of DRA along with Cl(-)/HCO(3)(-) exchange activity in tracheal epithelial cells 总被引:3,自引:0,他引:3
Wheat VJ Shumaker H Burnham C Shull GE Yankaskas JR Soleimani M 《American journal of physiology. Cell physiology》2000,279(1):C62-C71
Thickening of airway mucus and lungdysfunction in cystic fibrosis (CF) results, at least in part, fromabnormal secretion of Cl and HCO3across the tracheal epithelium. The mechanism of the defect in HCO3 secretion is ill defined; however, a lack ofapical Cl/HCO3 exchange may exist inCF. To test this hypothesis, we examined the expression ofCl/HCO3 exchangers in trachealepithelial cells exhibiting physiological features prototypical ofcystic fibrosis [CFT-1 cells, lacking a functional cystic fibrosistransmembrane conductance regulator (CFTR)] or normal trachea (CFT-1cells transfected with functional wild-type CFTR, termed CFT-WT). Cellswere grown on coverslips and were loaded with the pH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, andintracellular pH was monitored. Cl/HCO3exchange activity increased by ~300% in cells transfected with functional CFTR, with activities increasing from 0.034 pH/min in CFT-1cells to 0.11 in CFT-WT cells (P < 0.001, n = 8). This activity was significantly inhibited byDIDS. The mRNA expression of the ubiquitous basolateral AE-2Cl/HCO3 exchanger remained unchanged.However, mRNA encoding DRA, recently shown to be aCl/HCO3 exchanger (Melvin JE, Park K,Richardson L, Schultheis PJ, and Shull GE. J Biol Chem 274:22855-22861, 1999.) was abundantly expressed in cells expressingfunctional CFTR but not in cells that lacked CFTR or that expressedmutant CFTR. In conclusion, CFTR induces the mRNA expression of"downregulated in adenoma" (DRA) and, as a result, upregulates theapical Cl/HCO3 exchanger activity intracheal cells. We propose that the tracheal HCO3secretion defect in patients with CF is partly due to thedownregulation of the apical Cl/HCO3exchange activity mediated by DRA. 相似文献
6.
ANG II controls Na+-K+(NH+4)-2Cl- cotransport via 20-HETE and PKC in medullary thick ascending limb 总被引:1,自引:0,他引:1
Amlal Hassane; LeGoff Christian; Vernimmen Catherine; Soleimani Manoocher; Paillard Michel; Bichara Maurice 《American journal of physiology. Cell physiology》1998,274(4):C1047
Cell pH was monitored in medullary thick ascending limbs todetermine effects of ANG II onNa+-K+(NH+4)-2Clcotransport. ANG II at 1016to 1012 M inhibited30-50% (P < 0.005),but higher ANG II concentrations were stimulatory compared with the1012 M ANG II levelcotransport activity; eventually,106 M ANG II stimulated34% cotransport activity (P < 0.003). Inhibition by 1012M ANG II was abolished by phospholipase C (PLC), diacylglycerol lipase,or cytochrome P-450-dependentmonooxygenase blockade; 1012 M ANG II had no effectadditive to inhibition by 20-hydroxyeicosatetranoic acid (20-HETE).Stimulation by 106 M ANG IIwas abolished by PLC and protein kinase C (PKC) blockade and waspartially suppressed when the rise in cytosolicCa2+ was prevented. All ANG IIeffects were abolished by DUP-753 (losartan) but not by PD-123319. Thus1012 M ANG II inhibitsvia 20-HETE, whereas 5 × 1011 M ANG II stimulatesvia PKCNa+-K+(NH+4)-2Clcotransport; all ANG II effects involveAT1 receptors and PLC activation. 相似文献
7.
Beltinger J.; Hawkey C. J.; Stack W. A. 《American journal of physiology. Cell physiology》1999,276(4):C848
The effect of chronic exposure to transforming growth factor-(TGF-) on bradykinin-stimulated acute prostanoid production and ionsecretion in monolayers of HCA-7 colony 29 colonic epithelial cells hasbeen studied. Monolayers synthesized prostaglandinE2 (PGE2) at a basal rate of 2.10 ± 0.31 pg · monolayer1 · min1over 24 h. Bradykinin(108-105M) dose dependently increased acutePGE2 release by three orders ofmagnitude. This was associated with a rise in cAMP from 1.60 ± 0.14 to 2.90 ± 0.1 pmol/monolayer (P < 0.02) and a dose-dependent increase in short-circuit current (SCC).When monolayers were primed by a 24-h exposure to TGF-, basalPGE2 release rose to 6.31 ± 0.38 pg · monolayer1 · min1(TGF- concn 10 ng/ml; P = 0.001).However, the stimulation of acute prostaglandin release, intracellularcAMP, and increased SCC by bradykinin was significantly reduced bypreincubation with TGF-. Priming withPGE2(108-106M) over 24 h mimicked the effect of TGF- on bradykinin-induced changes in cAMP and SCC. These data suggest that enhanced chronic release of prostaglandins in response to stimulation with TGF- maydownregulate acute responses to bradykinin. In vivo, TGF- could havean important modulatory function in regulating secretion underinflammatory conditions. 相似文献
8.
PGE(2), Ca(2+), and cAMP mediate ATP activation of Cl(-) channels in pigmented ciliary epithelial cells 总被引:1,自引:0,他引:1
Fleischhauer JC Mitchell CH Peterson-Yantorno K Coca-Prados M Civan MM 《American journal of physiology. Cell physiology》2001,281(5):C1614-C1623
Purines regulate intraocular pressure. Adenosine activatesCl channels of nonpigmented ciliary epithelial cellsfacing the aqueous humor, enhancing secretion. Tamoxifen and ATPsynergistically activate Cl channels of pigmented ciliaryepithelial (PE) cells facing the stroma, potentially reducing netsecretion. The actions of nucleotides alone on Cl channelactivity of bovine PE cells were studied by electronic cell sorting,patch clamping, and luciferin/luciferase ATP assay. Clchannels were activated by ATP > UTP, ADP, and UDP, but not by 2-methylthio-ATP, all at 100 µM. UTP triggered ATP release. The second messengers Ca2+, prostaglandin (PG)E2,and cAMP activated Cl channels without enhancing effectsof 100 µM ATP. Buffering intracellular Ca2+activity with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acidor blocking PGE2 formation with indomethacininhibited ATP-triggered channel activation. The Rp stereoisomerof 8-bromoadenosine 3',5'-cyclic monophosphothioate inhibited proteinkinase A activity but mimicked 8-bromoadenosine 3',5'-cyclicmonophosphate. We conclude that nucleotides can act at >1 P2Yreceptor to trigger a sequential cascade involving Ca2+,PGE2, and cAMP. cAMP acts directly on Clchannels of PE cells, increasing stromal release and potentially reducing net aqueous humor formation and intraocular pressure. 相似文献
9.
Kang SH Vanden Berghe P Smith TK 《American journal of physiology. Cell physiology》2003,284(4):C839-C847
Whole cell patch-clamprecordings were made from cultured myenteric neurons taken from murineproximal colon. The micropipette contained Cs+ to removeK+ currents. Depolarization elicited a slowly activatingtime-dependent outward current (Itdo), whereasrepolarization was followed by a slowly deactivating tail current(Itail). Itdo andItail were present in ~70% of neurons. Weidentified these currents as Cl currents(ICl), because changing the transmembraneCl gradient altered the measured reversal potential(Erev) of both Itdo andItail with that for Itailshifted close to the calculated Cl equilibrium potential(ECl). ICl areCa2+-activated Cl current[ICl(Ca)] because they were Ca2+dependent. ECl, which was measured from theErev of ICl(Ca) using agramicidin perforated patch, was 33 mV. This value is more positivethan the resting membrane potential (56.3 ± 2.7 mV), suggestingmyenteric neurons accumulate intracellular Cl.-Conotoxin GIVA [0.3 µM; N-type Ca2+ channelblocker] and niflumic acid [10 µM; knownICl(Ca) blocker], decreased theICl(Ca). In conclusion, these neurons haveICl(Ca) that are activated by Ca2+entry through N-type Ca2+ channels. These currents likelyregulate postspike frequency adaptation. 相似文献
10.
Growth factorsstimulateNa+/H+exchange activity in many cell types but their effects on acidsecretion via this mechanism in renal tubules are poorly understood. Weexamined the regulation of HCO3absorption by nerve growth factor (NGF) in the rat medullary thickascending limb (MTAL), which absorbs HCO3via apical membraneNa+/H+exchange. MTAL were perfused in vitro with 25 mMHCO3 solutions (pH 7.4; 290 mosmol/kgH2O). Addition of 0.7 nMNGF to the bath decreased HCO3absorption from 13.1 ± 1.1 to 9.6 ± 0.8 pmol · min1 · mm1(P < 0.001). In contrast, with1010 M arginine vasopressin(AVP) in the bath, addition of NGF to the bath increasedHCO3 absorption from 8.0 ± 1.6 to12.5 ± 1.3 pmol · min1 · mm1(P < 0.01). Both effects of NGF wereblocked by genistein, consistent with the involvement of tyrosinekinase pathways. However, the AVP-dependent stimulation requiredactivation of protein kinase C (PKC), whereas the inhibition was PKCindependent, indicating that the NGF-induced signaling pathways leadingto inhibition and stimulation of HCO3absorption are distinct. Hypertonicity blocked the inhibition but notthe AVP-dependent stimulation, suggesting that hypertonicity and NGFmay inhibit HCO3 absorption via acommon mechanism. These data demonstrate that NGF inhibitsHCO3 absorption in the MTAL underbasal conditions but stimulates HCO3 absorption in the presence of AVP, effects that are mediated through distinct signal transduction pathways. They also show that AVP is acritical determinant of the response of the MTAL to growth factorstimulation and suggest that NGF can either inhibit or stimulateapical Na+/H+ exchange activitydepending on its interactions with other regulatory factors. Locallyproduced growth factors such as NGF may play a role in regulating renaltubule HCO3 absorption. 相似文献
11.
Apical and basolateral CO2-HCO-3 permeability in cultured bovine corneal endothelial cells 总被引:1,自引:0,他引:1
Bonanno Joseph A.; Guan Yi; Jelamskii Sergey; Kang Xiao Jun 《American journal of physiology. Cell physiology》1999,277(3):C545
Corneal endothelial function is dependent onHCO3 transport. However, the relativeHCO3 permeabilities of the apical andbasolateral membranes are unknown. Using changes in intracellular pHsecondary to removingCO2-HCO3 (at constant pH) or removing HCO3alone (at constant CO2) fromapical or basolateral compartments, we determined the relative apicaland basolateral HCO3 permeabilities and their dependencies on Na+ andCl. Removal ofCO2-HCO3from the apical side caused a steady-state alkalinization (+0.08 pHunits), and removal from the basolateral side caused an acidification(0.05 pH units). Removal ofHCO3 at constantCO2 indicated that the basolateralHCO3 fluxes were about three to fourtimes the apical fluxes. Reducing perfusateNa+ concentration to 10 mM had noeffect on apical flux but slowed basolateralHCO3 flux by one-half. In the absence of Cl, there was anapparent increase in apical HCO3 fluxunder constant-pH conditions; however, no net change could be measuredunder constant-CO2 conditions.Basolateral flux was slowed ~30% in the absence ofCl, but the net flux wasunchanged. The steady-state alkalinization after removal ofCO2-HCO3apically suggests that CO2diffusion may contribute to apicalHCO3 flux through the action of amembrane-associated carbonic anhydrase. Indeed, apicalCO2 fluxes were inhibited by theextracellular carbonic anhydrase inhibitor benzolamide and partiallyrestored by exogenous carbonic anhydrase. The presence ofmembrane-bound carbonic anhydrase (CAIV) was confirmed byimmunoblotting. We conclude that theNa+-dependent basolateralHCO3 permeability is consistent withNa+-nHCO3cotransport. Changes inHCO3 flux in the absence ofCl are most likely due toNa+-nHCO3cotransport-induced membrane potential changes that cannot bedissipated. Apical HCO3 permeabilityis relatively low, but may be augmented byCO2 diffusion in conjunction witha CAIV. 相似文献
12.
Müller-Ehmsen J Juvvadi P Thompson CB Tumyan L Croyle M Lingrel JB Schwinger RH McDonough AA Farley RA 《American journal of physiology. Cell physiology》2001,281(4):C1355-C1364
HumanNa+-K+-ATPase11,21, and 31heterodimers were expressed individually in yeast, and ouabainbinding and ATP hydrolysis were measured in membrane fractions. Theouabain equilibrium dissociation constant was 13-17 nM for11 and 31at 37°C and 32 nM for 21, indicatingthat the human -subunit isoforms have a similar high affinity forcardiac glycosides. K0.5 values for antagonism of ouabain binding by K+ were ranked in order as follows:2 (6.3 ± 2.4 mM) > 3(1.6 ± 0.5 mM) 1 (0.9 ± 0.6 mM),and K0.5 values for Na+ antagonismof ouabain binding to all heterodimers were 9.5-13.8 mM. Themolecular turnover for ATP hydrolysis by11 (6,652 min1) was abouttwice as high as that by 31 (3,145 min1). These properties of the human heterodimersexpressed in yeast are in good agreement with properties of the humanNa+-K+-ATPase expressed in Xenopusoocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-DHorisberger, L Lelievie, and K Geering. J Biol Chem275: 1976-1986, 2000). In contrast to Na+ pumpsexpressed in Xenopus oocytes, the21 complex in yeast membranes wassignificantly less stable than 11 or31, resulting in a lower functionalexpression level. The 21 complex was also more easily denatured by SDS than was the11 or the31 complex. 相似文献
13.
Characterization of norepinephrine-evoked inward currents in interstitial cells isolated from the rabbit urethra 总被引:6,自引:0,他引:6
Sergeant GP Thornbury KD McHale NG Hollywood MA 《American journal of physiology. Cell physiology》2002,283(3):C885-C894
Freshly dispersedinterstitial cells from the rabbit urethra were studied by using theperforated-patch technique. When cells were voltage clamped at 60 mVand exposed to 10 µM norepinephrine (NE) at 80-s intervals, eitherlarge single inward currents or a series of oscillatory inward currentsof diminishing amplitude were evoked. These currents were blocked byeither phentolamine (1 µM) or prazosin (1 µM), suggesting that theeffects of NE were mediated via 1-adrenoceptors.NE-evoked currents were depressed by the blockers ofCa2+-activated Cl currents, niflumic acid (10 µM), and 9-anthracenecarboxylic acid (9-AC, 1 mM). The reversalpotential of the above currents changed in a predictable manner whenthe Cl equilibrium potential was altered, againsuggesting that they were due to activation of a Clconductance. NE-evoked currents were decreased by 10 µM cyclopiazonic acid, suggesting that they were dependent on store-releasedCa2+. Inhibition of NE-evoked currents by the phospholipaseC inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate(100 µM) suggested that NE releases Ca2+ via an inositol1,4,5-trisphosphate (IP3)-dependent mechanism. Theseresults support the idea that stimulation of1-adrenoceptors releases Ca2+ from anIP3-sensitive store, which in turn activatesCa2+-activated Cl current in freshlydispersed interstitial cells of the rabbit urethra. This elevates slowwave frequency in these cells and may underlie the mechanismresponsible for increased urethral tone during nerve stimulation. 相似文献
14.
Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells 总被引:25,自引:0,他引:25
Chen L Wang L Zhu L Nie S Zhang J Zhong P Cai B Luo H Jacob TJ 《American journal of physiology. Cell physiology》2002,283(4):C1313-C1323
Patch-clamping and cell imageanalysis techniques were used to study the expression of thevolume-activated Cl current,ICl(vol), and regulatory volume decrease (RVD)capacity in the cell cycle in nasopharyngeal carcinoma cells (CNE-2Z). Hypotonic challenge caused CNE-2Z cells to swell and activated aCl current with a linear conductance, negligibletime-dependent inactivation, and a reversal potential close to theCl equilibrium potential. The sequence of anionpermeability was I > Br > Cl > gluconate. The Cl channelblockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),and ATP inhibited ICl(vol). Synchronous cultures of cells were obtained by the mitotic shake-off technique and by adouble chemical-block (thymidine and hydroxyurea) technique. Theexpression of ICl(vol) was cell cycle dependent,being high in G1 phase, downregulated in S phase, butincreasing again in M phase. Hypotonic solution activated RVD, whichwas cell cycle dependent and inhibited by the Cl channelblockers NPPB, tamoxifen, and ATP. The expression of ICl(vol) was closely correlated with the RVDcapacity in the cell cycle, suggesting a functional relationship.Inhibition of ICl(vol) by NPPB (100 µM)arrested cells in G0/G1. The data also suggest that expression of ICl(vol) and RVD capacity areactively modulated during the cell cycle. The volume-activatedCl current associated with RVD may therefore play animportant role during the cell cycle progress. 相似文献
15.
Functional and molecular characterization of an anion exchanger in airway serous epithelial cells 总被引:5,自引:0,他引:5
Loffing J Moyer BD Reynolds D Shmukler BE Alper SL Stanton BA 《American journal of physiology. Cell physiology》2000,279(4):C1016-C1023
Serous cells secreteCl and HCO3 and play an importantrole in airway function. Recent studies suggest that aCl/HCO3 anion exchanger (AE) maycontribute to Cl secretion by airway epithelial cells.However, the molecular identity, the cellular location, and thecontribution of AEs to Cl secretion in serous epithelialcells in tracheal submucosal glands are unknown. The goal of thepresent study was to determine the molecular identity, the cellularlocation, and the role of AEs in the function of serous epithelialcells. To this end, Calu-3 cells, a human airway cell line with aserous-cell phenotype, were studied by RT-PCR, immunoblot, andelectrophysiological analysis to examine the role of AEs inCl secretion. In addition, the subcellular location of AEproteins was examined by immunofluorescence microscopy. Calu-3 cellsexpressed mRNA and protein for AE2 as determined by RT-PCR and Westernblot analysis, respectively. Immunofluorescence microscopy identified AE2 in the basolateral membrane of Calu-3 cells in culture and rattracheal serous cells in situ. InCl/HCO3/Na+-containingmedia, the 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate(CPT-cAMP)-stimulated short-circuit anion current (Isc) was reduced by basolateral but not byapical application of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid(50 µM) and 4,4'-dinitrostilbene-2,2'-disulfonic acid [DNDS (500 µM)], inhibitors of AEs. In the absence of Na+ in thebath solutions, to eliminate the contributions of the Na+/HCO3 andNa+/K+/2Cl cotransporters toIsc, CPT-cAMP stimulated a small DNDS-sensitive Isc. Taken together with previous studies, theseobservations suggest that a small component of cAMP-stimulatedIsc across serous cells may be referable toCl secretion and that uptake of Cl acrossthe basolateral membrane may be mediated by AE2. 相似文献
16.
Griffin, M. Pamela. Role for anions in pulmonaryendothelial permeability. J. Appl.Physiol. 83(2): 615-622, 1997.-Adrenergic stimulation reduces albumin permeation across pulmonary artery endothelial monolayers and induces changes in cell morphology that aremediated by Cl flux. Wetested the hypothesis that anion-mediated changes in endothelial cellsresult in changes in endothelial permeability. We measured permeationof radiolabeled albumin across bovine pulmonary arterial endothelialmonolayers when the extracellular anion was Cl,Br,I,F, acetate(Ac), gluconate(G), and propionate(Pr). Permeability toalbumin (Palbumin)was calculated before and after addition of 0.2 mM of thephosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), whichreduces permeability. InCl, thePalbumin was 3.05 ± 0.86 × 106 cm/s andfell by 70% with the addition of IBMX. The initialPalbumin was lowest forPr andAc. InitialPalbumin was higher inBr,I,G, andF than inCl. A permeability ratiowas calculated to examine the IBMX effect. The greatest IBMX effect wasseen when Cl was theextracellular anion, and the order among halide anions wasCl > Br > I > F. Although the level ofextracellular Ca2+ concentration([Ca2+]o)varied over a wide range in the anion solutions,[Ca2+]odid not systematically affect endothelial permeability in this system.When Cl was theextracellular anion, varying[Ca2+]ofrom 0.2 to 2.8 mM caused a change in initialPalbumin but no changein the IBMX effect. The anion channel blockers4-acetamido-4-isothiocyanotostilbene-2,2-disulfonic acid(0.25 mM) and anthracene-9-carboxylic acid (0.5 mM) significantly altered initialPalbumin and the IBMXeffect. The anion transport blockers bumetanide (0.2 mM) and furosemide(1 mM) had no such effects. We conclude that extracellular anionsinfluence bovine pulmonary arterial endothelial permeability and thatthe pharmacological profile fits better with the activity of anionchannels than with other anion transport processes. 相似文献
17.
Ca(2+)-activated Cl(-) current in sheep lymphatic smooth muscle 总被引:1,自引:0,他引:1
Toland HM McCloskey KD Thornbury KD McHale NG Hollywood MA 《American journal of physiology. Cell physiology》2000,279(5):C1327-C1335
Freshly dispersed sheep mesenteric lymphaticsmooth muscle cells were studied at 37°C using the perforatedpatch-clamp technique with Cs+- and K+-filledpipettes. Depolarizing steps evoked currents that consisted ofL-type Ca2+ [ICa(L)]current and a slowly developing current. The slow current reversed at1 ± 1.5 mV with symmetrical Cl concentrationscompared with 23.2 ± 1.2 mV (n = 5) and34.3 ± 3.5 mV (n = 4) when externalCl was substituted with either glutamate (86 mM) orI (125 mM). Nifedipine (1 µM) blocked and BAY K 8644 enhanced ICa(L), the slow-developing sustainedcurrent, and the tail current. The Cl channel blockeranthracene-9-carboxylic acid (9-AC) reduced only the slowly developinginward and tail currents. Application of caffeine (10 mM) tovoltage-clamped cells evoked currents that reversed close to theCl equilibrium potential and were sensitive to 9-AC.Small spontaneous transient depolarizations and larger actionpotentials were observed in current clamp, and these were blocked by9-AC. Evoked action potentials were triphasic and had a prominentplateau phase that was selectively blocked by 9-AC. Similarly, fluidoutput was reduced by 9-AC in doubly cannulated segments ofspontaneously pumping sheep lymphatics, suggesting that theCa2+-activated Cl current plays an importantrole in the electrical activity underlying spontaneous activity in this tissue. 相似文献
18.
Peroxynitrite inhibits amiloride-sensitive Na+ currents in Xenopus oocytes expressing alpha beta gamma -rENaC 总被引:3,自引:0,他引:3
DuVall M. D.; Zhu S.; Fuller C. M.; Matalon S. 《American journal of physiology. Cell physiology》1998,274(5):C1417
We examined the effect of peroxynitrite(ONOO) on the cloned ratepithelial Na+ channel(-rENaC) expressed in Xenopusoocytes. 3-Morpholinosydnonimine (SIN-1) was used to concurrentlygenerate nitric oxide (· NO) and superoxide(O2 ·), which react toform ONOO, a species knownto promote protein nitration and oxidation. Under control conditions,oocytes displayed an amiloride-sensitive whole cell conductance of 7.4 ± 2.8 (SE) µS. When incubated at 18°C with SIN-1 (1 mM) for 2 h (final ONOO concentration = 10 µM), the amiloride-sensitive conductance was reduced to0.8 ± 0.5 µS. To evaluate whether the observed inhibition was due to ONOO, as opposedto · NO, we also exposed oocytes to SIN-1 in the presence ofurate (500 µM), a scavenger ofONOO and superoxidedismutase, which scavengesO2 ·, converting SIN-1from an ONOO to an· NO donor. Under these conditions, conductance values remained at control levels following SIN-1 treatment.Tetranitromethane, an agent that oxidizes sulfhydryl groups at pH6, also inhibited the amiloride-sensitive conductance. These datasuggest that oxidation of critical sulfhydryl groups within rENaC byONOO directly inhibitschannel activity. 相似文献
19.
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
20.
Shuba YM Prevarskaya N Lemonnier L Van Coppenolle F Kostyuk PG Mauroy B Skryma R 《American journal of physiology. Cell physiology》2000,279(4):C1144-C1154
Patch-clamp recordings were used to study ioncurrents induced by cell swelling caused by hypotonicity in humanprostate cancer epithelial cells, LNCaP. The reversal potential of the swelling-evoked current suggested that Cl was the primarycharge carrier (termed ICl,swell). Theselectivity sequence of the underlying volume-regulated anion channels(VRACs) for different anions wasBrI > Cl > F > methanesulfonate glutamate, with relativepermeability numbers of 1.26, 1.20, 1.0, 0.77, 0.49, and 0.036, respectively. The current-voltage patterns of the whole cell currentsas well as single-channel currents showed moderate outwardrectification. Unitary VRAC conductance was determined at 9.6 ± 1.8 pS. Conventional Cl channel blockers5-nitro-2-(3-phenylpropylamino)benzoic acid (100 µM) and DIDS (100 µM) inhibited whole cell ICl,swell in a voltage-dependent manner, with the block decreasing from 39.6 ± 9.7% and 71.0 ± 11.0% at +50 mV to 26.2 ± 7.2% and14.5 ± 6.6% at 100 mV, respectively. Verapamil (50 µM), astandard Ca2+ antagonist and P-glycoprotein functioninhibitor, depressed the current by a maximum of 15%. Protein tyrosinekinase inhibitors downregulated ICl,swell(genistein with an IC50 of 2.6 µM and lavendustin A by60 ± 14% at 1 µM). The protein tyrosine phosphatase inhibitorsodium orthovanadate (500 µM) stimulatedICl,swell by 54 ± 11%. We conclude thatVRACs in human prostate cancer epithelial cells are modulated viaprotein tyrosine phosphorylation. 相似文献