共查询到20条相似文献,搜索用时 31 毫秒
1.
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
2.
Xiao GQ Qu Y Sun ZQ Mochly-Rosen D Boutjdir M 《American journal of physiology. Cell physiology》2001,281(5):C1477-C1486
Investigation of the role ofindividual protein kinase C (PKC) isozymes in the regulation ofNa+ channels has been largely limited by the lack ofisozyme-selective modulators. Here we used a novel peptide-specificactivator (V1-7) of PKC and other peptide isozyme-specificinhibitors in addition to the general PKC activator phorbol12-myristate 13-acetate (PMA) to dissect the role of individual PKCs inthe regulation of the human cardiac Na+ channel hH1,heterologously expressed in Xenopus oocytes. Peptides wereinjected individually or in combination into the oocyte. Whole cellNa+ current (INa) was recorded usingtwo-electrode voltage clamp. V1-7 (100 nM) and PMA (100 nM)inhibited INa by 31 ± 5% and 44 ± 8% (at 20 mV), respectively. These effects were not seen with thescrambled peptide for V1-7 (100 nM) or the PMA analog4-phorbol 12,13-didecanoate (100 nM). However, V1-7-and PMA-induced INa inhibition was abolished byV1-2, a peptide-specific antagonist of PKC. Furthermore,PMA-induced INa inhibition was not altered by100 nM peptide-specific inhibitors for -, -, -, or PKC. PMAand V1-7 induced translocation of PKC from soluble toparticulate fraction in Xenopus oocytes. This translocationwas antagonized by V1-2. In native rat ventricular myocytes,PMA and V1-7 also inhibited INa; thisinhibition was antagonized by V1-2. In conclusion, the resultsprovide evidence for selective regulation of cardiac Na+channels by PKC isozyme. 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.
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
5.
Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms 总被引:3,自引:0,他引:3
Song Jaekyung Cecilia; Hanson Celina M.; Tsai Vance; Farokhzad Omid C.; Lotz Margaret; Matthews Jeffrey B. 《American journal of physiology. Cell physiology》2001,281(2):C649
The phorbol ester phorbol12-myristate 13-acetate (PMA) inhibits Cl secretion(short-circuit current, Isc) and decreasesbarrier function (transepithelial resistance, TER) in T84 epithelia. To elucidate the role of specific protein kinase C (PKC) isoenzymes inthis response, we compared PMA with two non-phorbol activators of PKC(bryostatin-1 and carbachol) and utilized three PKC inhibitors (Gö-6850, Gö-6976, and rottlerin) with different isozymeselectivity profiles. PMA sequentially inhibited cAMP-stimulatedIsc and decreased TER, as measured byvoltage-current clamp. By subcellular fractionation and Western blot,PMA (100 nM) induced sequential membrane translocation of the novelPKC followed by the conventional PKC and activated both isozymesby in vitro kinase assay. PKC was activated by PMA but did nottranslocate. By immunofluorescence, PKC redistributed to thebasolateral domain in response to PMA, whereas PKC moved apically.Inhibition of Isc by PMA was prevented by theconventional and novel PKC inhibitor Gö-6850 (5 µM) but not theconventional isoform inhibitor Gö-6976 (5 µM) or the PKCinhibitor rottlerin (10 µM), implicating PKC in inhibition ofCl secretion. In contrast, both Gö-6976 andGö-6850 prevented the decline of TER, suggesting involvement ofPKC. Bryostatin-1 (100 nM) translocated PKC and PKC andinhibited cAMP-elicited Isc. However, unlikePMA, bryostatin-1 downregulated PKC protein, and the decrease in TERwas only transient. Carbachol (100 µM) translocated only PKC andinhibited Isc with no effect on TER. Gö-6850 but not Gö-6976 or rottlerin blocked bryostatin-1and carbachol inhibition of Isc. We concludethat basolateral translocation of PKC inhibits Clsecretion, while apical translocation of PKC decreases TER. Thesedata suggest that epithelial transport and barrier function can bemodulated by distinct PKC isoforms. 相似文献
6.
Li Yansui; Kuang Kunyan; Yerxa Benjamin; Wen Quan; Rosskothen Heinz; Fischbarg Jorge 《American journal of physiology. Cell physiology》2001,281(2):C595
Rabbit conjunctival epithelium exhibits UTP-dependentCl secretion into the tears. We investigated whetherfluid secretion also takes place. Short-circuit current(Isc) was 14.9 ± 1.4 µA/cm2(n = 16). Four P2Y2 purinergic receptoragonists [UTP and the novel compounds INS365, INS306, and INS440(Inspire Pharmaceuticals)] added apically (10 µM) resulted intemporary (~30 min) Isc increases (88%, 66%,57%, and 28%, respectively; n = 4 each). Importantly, the conjunctiva transported fluid from serosa to mucosa at a rate of6.5 ± 0.7 µl · h1 · cm2 (range2.1-15.3, n = 20). Fluid transport was stimulatedby mucosal additions of 10 µM: 1) UTP, from 7.4 ± 2.3 to 10.7 ± 3.3 µl · h1 · cm2,n = 5; and 2) INS365, from 6.3 ± 1.0 to 9.8 ± 2.5 µl · h1 · cm2,n = 5. Fluid transport was abolished by 1 mMouabain (n = 5) and was drastically inhibited by 300 µM quinidine (from 6.4 ± 1.2 to 3.6 ± 1.0 µl · h1 · cm2,n = 4). We conclude that this epithelium secretes fluidactively and that P2Y2 agonists stimulate bothCl and fluid secretions. 相似文献
7.
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
8.
Buhagiar Kerrie A.; Hansen Peter S.; Bewick Nerida L.; Rasmussen Helge H. 《American journal of physiology. Cell physiology》2001,281(3):C1059
A reduction in angiotensinII (ANG II) in vivo by treatment of rabbits with theangiotensin-converting enzyme inhibitor, captopril, increasesNa+-K+ pump current (Ip)of cardiac myocytes. This increase is abolished by exposure of myocytesto ANG II in vitro. Because ANG II induces translocation of the-isoform of protein kinase C (PKC), we examined whether thisisozyme regulates the pump. We treated rabbits with captopril, isolatedmyocytes, and measured Ip of myocytes voltageclamped with wide-tipped patch pipettes. Ip ofmyocytes from captopril-treated rabbits was larger thanIp of myocytes from controls. ANG II superfusionof myocytes from captopril-treated rabbits decreasedIp to levels similar to controls. Inclusion ofPKC-specific blocking peptide in pipette solutions used to perfusethe intracellular compartment abolished the effect of ANG II. Inclusionof RACK, a PKC-specific activating peptide, in pipettesolutions had an effect on Ip that was similarto that of ANG II. There was no additive effect of ANG II andRACK. We conclude that PKC regulates the sarcolemmalNa+-K+ pump. 相似文献
9.
TGF-beta effects on epithelial ion transport and barrier: reduced Cl- secretion blocked by a p38 MAPK inhibitor 总被引:1,自引:0,他引:1
Growthfactors affect a variety of epithelial functions. We examined theability of TGF- to modulate epithelial ion transport andpermeability. Filter-grown monolayers of human colonic epithelia, T84and HT-29 cells, were treated with TGF- (0.1-100 ng/ml,15 min-72 h) or infected with an adenoviral vector encodingTGF- (Ad-TGF) for 144 h. Ion transport (i.e., short-circuitcurrent, Isc) and transepithelial resistance(TER) were assessed in Ussing chambers. Neither recombinant TGF- norAd-TGF infection affected baseline Isc;however, exposure to 1 ng/ml TGF- led to a significant (30-50%) reduction in the Isc responses toforskolin, vasoactive intestinal peptide, and cholera toxin (agentsthat evoke Cl secretion via cAMP mobilization) and to thecell-permeant dibutyryl cAMP. Pharmacological analysis of signalingpathways revealed that the inhibition of cAMP-driven epithelialCl secretion by TGF- was blocked by pretreatment withSB-203580, a specific inhibitor of p38 MAPK, but not by inhibitors ofJNK, ERK1/2 MAPK, or phosphatidylinositol 3'-kinase. TGF- enhanced the barrier function of the treated monolayers by up to threefold asassessed by TER; however, this event was temporally displaced from thealtered Isc response, being statisticallysignificant only at 72 h posttreatment. Thus, in addition toTGF- promotion of epithelial barrier function, we show that thisgrowth factor also reduces responsiveness to cAMP-dependentsecretagogues in a chronic manner and speculate that this serves as abraking mechanism to limit secretory enteropathies. 相似文献
10.
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
11.
Alterations in airway ion transport in NKCC1-deficient mice 总被引:2,自引:0,他引:2
Grubb B. R.; Pace A. J.; Lee E.; Koller B. H.; Boucher R. C. 《American journal of physiology. Cell physiology》2001,281(2):C615
Airways of Na+-K+-2Cl(NKCC1)-deficient mice (/) were studied in Ussing chambers todetermine the role of the basolateral NKCC1 in transepithelial anionsecretion. The basal short-circuit current (Isc)of tracheae and bronchi from adult mice did not differ betweenNKCC1/ and normal mice, whereas NKCC1/ tracheae from neonatalmice exhibited a significantly reduced basalIsc. In normal mouse tracheae, sensitivity tothe NKCC1 inhibitor bumetanide correlated inversely with the age of themouse. In contrast, tracheae from NKCC1/ mice at all ages wereinsensitive to bumetanide. The anion secretory response to forskolindid not differ between normal and NKCC1/ tissues. However, whenlarger anion secretory responses were induced with UTP, airways fromthe NKCC1/ mice exhibited an attenuated response. Ion substitutionand drug treatment protocols suggested that HCO secretion compensated for reduced Cl secretion inNKCC1/ airway epithelia. The absence of spontaneous airway diseaseor pathology in airways from the NKCC1/ mice suggests that theNKCC1 mutant mice are able to compensate adequately for absence of theNKCC1 protein. 相似文献
12.
Regulation of ATP-sensitive K(+) channels by protein kinase C in murine colonic myocytes 总被引:1,自引:0,他引:1
Jun JY Kong ID Koh SD Wang XY Perrino BA Ward SM Sanders KM 《American journal of physiology. Cell physiology》2001,281(3):C857-C864
We investigated the regulation ofATP-sensitive K+ (KATP) currents in murinecolonic myocytes with patch-clamp techniques. Pinacidil(105 M) activated inward currents in the presence of highexternal K+ (90 mM) at a holding potential of 80 mV indialyzed cells. Glibenclamide (105 M) suppressedpinacidil-activated current. Phorbol 12,13-dibutyrate (PDBu; 2 × 107 M) inhibited pinacidil-activated current.4--Phorbol ester (5 × 107 M), an inactive formof PDBu, had no effect on pinacidil-activated current. In cell-attachedpatches, the open probability of KATP channels wasincreased by pinacidil, and PDBu suppressed openings ofKATP channels. When cells were pretreated withchelerythrine (106 M) or calphostin C (107M), inhibition of the pinacidil-activated whole cell currents by PDBuwas significantly reduced. In cells studied with the perforated patchtechnique, PDBu also inhibited pinacidil-activated current, and thisinhibition was reduced by chelerythrine (106 M).Acetylcholine (ACh; 105 M) inhibited pinacidil-activatedcurrents, and preincubation of cells with calphostin C(107 M) decreased the effect of ACh. Cells dialyzed withprotein kinase C -isoform (PKC) antibody had normal responses topinacidil, but the effects of PDBu and ACh on KATP wereblocked in these cells. Immunofluorescence and Western blots showedexpression of PKC in intact muscles and isolated smooth muscle cellsof the murine proximal colon. These data suggest that PKC regulates KATP in colonic muscle cells and that the effects of ACh onKATP are largely mediated by PKC. PKC appears to be themajor isozyme that regulates KATP in murine colonic myocytes. 相似文献
13.
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
14.
Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells 总被引:23,自引:0,他引:23
Schneider Eric; Schmid-Kotsas Alexandra; Zhao Jinshun; Weidenbach Hans; Schmid Roland M.; Menke Andre; Adler Guido; Waltenberger Johannes; Grunert Adolf; Bachem Max G. 《American journal of physiology. Cell physiology》2001,281(2):C532
The aim of thisstudy was to identify fibrogenic mediators stimulatingactivation, proliferation, and/or matrix synthesis of rat pancreaticstellate cells (PSC). PSC were isolated from the pancreas of normalWistar rats and from rats with cerulein pancreatitis. Cell activationwas demonstrated by immunofluorescence microscopy of smooth muscle-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin,and transforming growth factor (TGF)-1. Proliferationwas measured by bromodeoxyuridine incorporation. Matrix synthesis wasdemonstrated on the protein and mRNA level. Within a few days inprimary culture, PSC changed their phenotype from fat-storing toSMA-positive myofibroblast-like cells expressing platelet-derivedgrowth factor (PDGF) - and PDGF -receptors. TGF-1and tumor necrosis factor (TNF)- accelerated the change in thecells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basicfibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 ± 0.49- and 2.96 ± 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 ± 1.13-fold), 5 ng/ml TGF-1 (2.46 ± 0.89-fold), 20 ng/ml PDGF (2.27 ± 0.68-fold), and 50 ng/ml TGF- (1.87 ± 0.19-fold). As shownby RT-PCR, PSC express predominantly the splice variant EIII-A offibronectin. Immunofluorescence microscopy and Northern blot confirmedthat in particular bFGF and TGF-1 stimulated thesynthesis of fibronectin and collagens type I and III. In conclusion,our data demonstrate that 1) TGF-1 andTNF- accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF,TGF-1, PDGF, and, to a lesser extent, TGF- stimulateextracellular matrix synthesis of cultured rat PSC. 相似文献
15.
Arachidonic acid mediates dual effect of TNF-alpha on Ca2+ transients and contraction of adult rat cardiomyocytes 总被引:1,自引:0,他引:1
Amadou A Nawrocki A Best-Belpomme M Pavoine C Pecker F 《American journal of physiology. Cell physiology》2002,282(6):C1339-C1347
Tumor necrosisfactor (TNF)- has a biphasic effect on heart contractility andstimulates phospholipase A2 (PLA2) incardiomyocytes. Because arachidonic acid (AA) exerts a dual effect onintracellular Ca2+ concentration([Ca2+]i) transients, we investigated thepossible role of AA as a mediator of TNF- on[Ca2+]i transients and contraction withelectrically stimulated adult rat cardiac myocytes. At a lowconcentration (10 ng/ml) TNF- produced a 40% increase in theamplitude of both [Ca2+]i transients andcontraction within 40 min. At a high concentration (50 ng/ml) TNF-evoked a biphasic effect comprising an initial positive effect peakingat 5 min, followed by a sustained negative effect leading to50-40% decreases in [Ca2+]i transientsand contraction after 30 min. Both the positive and negative effects ofTNF- were reproduced by AA and blocked by arachidonyltrifluoromethylketone (AACOCF3), an inhibitor of cytosolic PLA2.Lipoxygenase and cyclooxygenase inhibitors reproduced the high-doseeffects of TNF- and AA. The negative effects of TNF- and AA werealso reproduced by sphingosine and were abrogated by the ceramidaseinhibitor n-oleoylethanolamine. These results point out thekey role of the cytosolic PLA2/AA pathway in mediating thecontractile effects of TNF-. 相似文献
16.
Relationship between intracellular pH and chloride in Xenopus oocytes expressing the chloride channel ClC-0 总被引:1,自引:0,他引:1
During maturation of oocytes,Cl conductance (GCl) oscillatesand intracellular pH (pHi) increases. ElevatingpHi permits the protein synthesis essential to maturation.To examine whether changes in GCl andpHi are coupled, the Cl channel ClC-0 washeterologously expressed. Overexpressing ClC-0 elevatespHi, decreases intracellular Cl concentration([Cl]i), and reduces volume. Acuteacidification with butyrate does not activate acid extrusion inClC-0-expressing or control oocytes. The ClC-0-induced pHichange increases after overnight incubation at extracellular pH 8.5 butis unaltered after incubation at extracellular pH 6.5. Membranedepolarization did not change pHi. In contrast, hyperpolarization elevates pHi. Thus neither membranedepolarization nor acute activation of acid extrusion accounts for theClC-0-dependent alkalinization. Overnight incubation in lowextracellular Cl concentration increases pHiand decreases [Cl]i in control and ClC-0expressing oocytes, with the effect greater in the latter. Incubationin hypotonic, low extracellular Cl solutions preventedpHi elevation, although the decrease in[Cl]i persisted. Taken together, ourobservations suggest that KCl loss leads to oocyte shrinkage, whichtransiently activates acid extrusion. In conclusion, expressing ClC-0in oocytes increases pHi and decreases[Cl]i. These parameters are coupled viashrinkage activation of proton extrusion. Normal, cyclical changes ofoocyte GCl may exert an effect onpHi via shrinkage, thus inducing meiotic maturation. 相似文献
17.
Knepler JL Taher LN Gupta MP Patterson C Pavalko F Ober MD Hart CM 《American journal of physiology. Cell physiology》2001,281(3):C1064-C1075
Nitric oxide (·NO) attenuates hydrogen peroxide(H2O2)-mediated barrier dysfunction in culturedporcine pulmonary artery endothelial cells (PAEC) (Gupta MP, Ober MD,Patterson C, Al-Hassani M, Natarajan V, and Hart, CM. Am JPhysiol Lung Cell Mol Physiol 280: L116-L126, 2001). However,·NO rapidly combines with superoxide (O ) to formthe powerful oxidant peroxynitrite (ONOO), which wehypothesized would cause PAEC monolayer barrier dysfunction. To testthis hypothesis, we treated PAEC with ONOO (500 µM) or3-morpholinosydnonimine hydrochloride (SIN-1; 1-500 µM).SIN-1-mediated ONOO formation was confirmed by monitoringthe oxidation of dihydrorhodamine 123 to rhodamine. BothONOO and SIN-1 increased albumin clearance(P < 0.05) in the absence of cytotoxicity and alteredthe architecture of the cytoskeletal proteins actin and -catenin asdetected by immunofluorescent confocal imaging.ONOO-induced barrier dysfunction was partially reversibleand was attenuated by cysteine. Both ONOO and SIN-1nitrated tyrosine residues, including those on -catenin and actin,and oxidized proteins in PAEC. The introduction of actin treated withONOO into PAEC monolayers via liposomes alsoresulted in barrier dysfunction. These results indicate thatONOO directly alters endothelial cytoskeletal proteins,leading to barrier dysfunction. 相似文献
18.
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
19.
Blaug Sasha; Hybiske Kevin; Cohn Jonathan; Firestone Gary L.; Machen Terry E.; Miller Sheldon S. 《American journal of physiology. Cell physiology》2001,281(2):C633
Mammary epithelial 31EG4 cells (MEC) were grown as monolayers onfilters to analyze the apical membrane mechanisms that help mediate ionand fluid transport across the epithelium. RT-PCR showed the presenceof cystic fibrosis transmembrane conductance regulator (CFTR) andepithelial Na+ channel (ENaC) message, and immunomicroscopyshowed apical membrane staining for both proteins. CFTR was alsolocalized to the apical membrane of native human mammary ductepithelium. In control conditions, mean values of transepithelialpotential (apical-side negative) and resistance(RT) are 5.9 mV and 829 · cm2, respectively. The apical membranepotential (VA) is 40.7 mV, and the mean ratioof apical to basolateral membrane resistance (RA/RB) is 2.8. Apicalamiloride hyperpolarized VA by 19.7 mV andtripled RA/RB. AcAMP-elevating cocktail depolarized VA by 17.6 mV, decreased RA/RB by60%, increased short-circuit current by 6 µA/cm2,decreased RT by 155 · cm2, and largely eliminated responses toamiloride. Whole cell patch-clamp measurements demonstratedamiloride-inhibited Na+ currents [linear current-voltage(I-V) relation] and forskolin-stimulated Clcurrents (linear I-V relation). A capacitance probe methodshowed that in the control state, MEC monolayers either absorbed orsecreted fluid (2-4µl · cm2 · h1). Fluidsecretion was stimulated either by activating CFTR (cAMP) or blockingENaC (amiloride). These data plus equivalent circuit analysis showedthat 1) fluid absorption across MEC is mediated byNa+ transport via apical membrane ENaC, and fluid secretionis mediated, in part, by Cl transport via apicalCFTR; 2) in both cases, appropriate counterions move throughtight junctions to maintain electroneutrality; and 3)interactions among CFTR, ENaC, and tight junctions allow MEC to eitherabsorb or secrete fluid and, in situ, may help control luminal[Na+] and [Cl]. 相似文献
20.
Agonist-specific cross talk between ERKs and p38(mapk) regulates PGI(2) synthesis in endothelium 总被引:1,自引:0,他引:1
Houliston RA Pearson JD Wheeler-Jones CP 《American journal of physiology. Cell physiology》2001,281(4):C1266-C1276
We have examined the mechanisms regulatingprostacyclin (PGI2) synthesis after acute exposure of humanumbilical vein endothelial cells (HUVEC) to interleukin-1 (IL-1).IL-1 evoked an early (30 min) release of PGI2 and[3H]arachidonate that was blocked by the cytosolicphospholipase A2 (cPLA2) inhibitorarachidonyl trifluoromethyl ketone. IL-1-mediated activationof extracellular signal-regulated kinase 1/2 (ERK1/2; p42/p44mapk) coincided temporally with phosphorylation ofcPLA2 and with the onset of PGI2synthesis. The mitogen-activated protein kinase (MAPK) kinase (MEK)inhibitors, PD-98059 and U-0126, blocked IL-1-induced ERKactivation and partially attenuated cPLA2phosphorylation and PGI2 release, suggesting thatERK-dependent and -independent pathways regulate cPLA2phosphorylation. SB-203580 treatment enhanced IL-1-induced MEK,p42/44mapk, and cPLA2 phosphorylation butreduced thrombin-stimulated MEK and p42/44mapk activation.IL-1, but not thrombin, activated Raf-1 as assessed byimmune-complex kinase assay, as did SB-203580 alone. These results showthat IL-1 causes an acute upregulation of PGI2generation in HUVEC, establish a role for theMEK/ERK/cPLA2 pathway in this early release, and provideevidence for an agonist-specific cross talk between p38mapkand p42/44mapk that may reflect receptor-specificdifferences in the signaling elements proximal to MAPK activation. 相似文献