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1.
Protein kinase C mediates the inhibitory effect of substance P on HCO3- secretion from guinea pig pancreatic ducts 总被引:1,自引:0,他引:1
Hegyi P Rakonczay Z Tiszlavicz L Varró A Tóth A Rácz G Varga G Gray MA Argent BE 《American journal of physiology. Cell physiology》2005,288(5):C1030-C1041
The inhibitory control of pancreatic ductal HCO3 secretion may be physiologically important in terms of limiting the hydrostatic pressure developed within the ducts and in terms of switching off pancreatic secretion after a meal. Substance P (SP) inhibits secretin-stimulated HCO3 secretion by modulating a Cl-dependent HCO3 efflux step at the apical membrane of the duct cell (Hegyi P, Gray MA, and Argent BE. Am J Physiol Cell Physiol 285: C268C276, 2003). In the present study, we have shown that SP is present in periductal nerves within the guinea pig pancreas, that PKC mediates the effect of SP, and that SP inhibits an anion exchanger on the luminal membrane of the duct cell. Secretin (10 nM) stimulated HCO3 secretion by sealed, nonperfused, ducts about threefold, and this effect was totally inhibited by SP (20 nM). Phorbol 12,13-dibutyrate (PDBu; 100 nM), an activator of PKC, reduced basal HCO3 secretion by 40% and totally blocked secretin-stimulated secretion. In addition, bisindolylmaleimide I (1 nM to 1 µM), an inhibitor of PKC, relieved the inhibitory effect of SP on secretin-stimulated HCO3 secretion and also reversed the inhibitory effect of PDBu. Western blot analysis revealed that guinea pig pancreatic ducts express the -, I-, -, -, -, -, -, and µ-isoforms of PKC. In microperfused ducts, luminal H2DIDS (0.5 mM) caused intracellular pH to alkalinize and, like SP, inhibited basal and secretin-stimulated HCO3 secretion. SP did not inhibit secretion further when H2DIDS was present in the lumen, suggesting that SP and H2DIDS both inhibit the activity of an anion exchanger on the luminal membrane of the duct cell. pancreas; Cl/HCO3 exchanger; inhibition; epithelium 相似文献
2.
Uezono Y Kaibara M Murasaki O Taniyama K 《American journal of physiology. Cell physiology》2004,287(4):C885-C894
We studied the functions of -subunits of Gi/o protein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl currents in oocytes expressing 2-adrenoceptor and the protein kinase A-dependent Cl channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 5-Hydroxytryptamine (5-HT), [D-Ala2, D-Leu5]-enkephalin (DADLE), and baclofen enhanced ISO-induced cAMP levels and CFTR currents in oocytes expressing 2-adrenoceptor-CFTR and 5-HT1A receptor (5-HT1AR), -opioid receptor, or GABAB receptor, respectively. 5-HT also enhanced pituitary adenylate cyclase activating peptide (PACAP) 38-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor, CFTR and 5-HT1AR. The 5-HT-induced enhancement of Gs-coupled receptor-mediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin (Gt). The 5-HT-induced enhancement was further augmented by coexpression of the G-activated form of adenylate cyclase (AC) type II but not AC type III. Thus -subunits of Gi/o protein contribute to the enhancement of Gs-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT1AR or -opioid receptor alone. They elicited Ca2+-activated Cl currents in oocytes coexpressing these receptors with the G-activated form of phospholipase C (PLC)-2 but not with PLC-1. These currents were inhibited by pretreatment with PTX and coexpression of Gt, suggesting that -subunits of Gi/o protein activate PLC-2 and then cause intracellular Ca2+ mobilization. Our results indicate that -subunits of Gi/o protein participate in diverse intracellular signals, enhancement of Gs-coupled receptor-mediated responses, and intracellular Ca2+ mobilization. G protein-coupled receptor; cystic fibrosis transmembrane conductance regulator gene; cross talk; electrophysiology 相似文献
3.
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
4.
Similarity of A(3)-adenosine and swelling-activated Cl(-) channels in nonpigmented ciliary epithelial cells 总被引:1,自引:0,他引:1
Carré DA Mitchell CH Peterson-Yantorno K Coca-Prados M Civan MM 《American journal of physiology. Cell physiology》2000,279(2):C440-C451
Chloride release from nonpigmented ciliary epithelial (NPE)cells is a final step in forming aqueous humor, and adenosine stimulates Cl transport by these cells. Whole cell patchclamping of cultured human NPE cells indicated that theA3-selective agonist1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl--D-ribofuranuronamide (IB-MECA) stimulated currents (IIB-MECA) by~90% at +80 mV. Partial replacement of external Clwith aspartate reduced outward currents and shifted the reversal potential (Vrev) from 23 ± 2 mV to0.0 ± 0.7 mV. Nitrate substitution had little effect. Perfusionwith the Cl channel blockers5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acidinhibited the currents. Partial Cl replacement withaspartate and NO3, and perfusion with NPPB, hadsimilar effects on the swelling-activated whole cell currents(ISwell). Partial cyclamate substitution for external Cl inhibited inward and outward currents of bothIIB-MECA and ISwell. Bothsets of currents also showed outward rectification and inactivation atlarge depolarizing potentials. The results are consistent with theconcept that A3-subtype adenosine agonists and swellingactivate a common population of Cl channels. 相似文献
5.
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
6.
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
7.
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
8.
Impaired [Ca(2+)](i) and pH(i) responses to kappa-opioid receptor stimulation in the heart of chronically hypoxic rats 总被引:1,自引:0,他引:1
Pei JM Zhou JJ Bian JS Yu XC Fung ML Wong TM 《American journal of physiology. Cell physiology》2000,279(5):C1483-C1494
-Opioid receptor (-OR)stimulation with U50,488H, a selective -OR agonist, or activation ofprotein kinase C (PKC) with 4-phorbol 12-myristate 13-acetate (PMA), anactivator of PKC, decreased the electrically induced intracellularCa2+ ([Ca2+]i) transient andincreased the intracellular pH (pHi) in single ventricularmyocytes of rats subjected to 10% oxygen for 4 wk. The effects ofU50,488H were abolished by nor-binaltorphimine, a selective -ORantagonist, and calphostin C, a specific inhibitor of PKC, while theeffects of PMA were abolished by calphostin C andethylisopropylamiloride (EIPA), a potent Na+/H+exchange blocker. In both right hypertrophied and leftnonhypertrophied ventricles of chronically hypoxic rats, the effects ofU50,488H or PMA on [Ca2+]i transient andpHi were significantly attenuated and completely abolished,respectively. Results are first evidence that the[Ca2+]i and pHi responses to-OR stimulation are attenuated in the chronically hypoxic rat heart,which may be due to reduced responses to PKC activation. Responses toall treatments were the same for right and left ventricles, indicatingthat the functional impairment is independent of hypertrophy. -ORmRNA expression was the same in right and left ventricles of bothnormoxic and hypoxic rats, indicating no regional specificity. 相似文献
9.
Maglova Lilia M.; Crowe William E.; Smith Peter R.; Altamirano Anibal A.; Russell John M. 《American journal of physiology. Cell physiology》1998,275(5):C1330
We examined the effects of human cytomegalovirus (HCMV)infection on theNa+-K+-Clcotransporter (NKCC) in a human fibroblast cell line. Using the Cl-sensitive dye MQAE, weshowed that the mock-infected MRC-5 cells express a functional NKCC.1) IntracellularCl concentration([Cl]i)was significantly reduced from 53.4 ± 3.4 mM to 35.1 ± 3.6 mMfollowing bumetanide treatment. 2)Net Cl efflux caused byreplacement of external Clwith gluconate was bumetanide sensitive.3) InCl-depleted mock-infectedcells, the Cl reuptake rate(in HCO3-free media) was reduced inthe absence of external Na+ and bytreatment with bumetanide. After HCMV infection, we found that although[Cl]iincreased progressively [24 h postexposure (PE), 65.2 ± 4.5 mM; 72 h PE, 80.4 ± 5.0 mM], the bumetanide andNa+ sensitivities of[Cl]iand net Cl uptake and losswere reduced by 24 h PE and abolished by 72 h PE. Western blots usingthe NKCC-specific monoclonal antibody T4 showed an approximatelyninefold decrease in the amount of NKCC protein after 72 h ofinfection. Thus HCMV infection resulted in the abolition of NKCCfunction coincident with the severe reduction in the amount of NKCCprotein expressed. 相似文献
10.
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
11.
Dynamic regulation of Na(+)-K(+)-2Cl(-) cotransporter surface expression by PKC-{epsilon} in Cl(-)--secretory epithelia 总被引:1,自引:0,他引:1
Del Castillo IC Fedor-Chaiken M Song JC Starlinger V Yoo J Matlin KS Matthews JB 《American journal of physiology. Cell physiology》2005,289(5):C1332-C1342
In secretory epithelia, activation of PKC by phorbol ester and carbachol negatively regulates Cl secretion, the transport event of secretory diarrhea. Previous studies have implicated the basolateral Na+-K+-2Cl cotransporter (NKCC1) as a target of PKC-dependent inhibition of Cl secretion. In the present study, we examined the regulation of surface expression of NKCC1 in response to the activation of PKC. Treatment of confluent T84 intestinal epithelial cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA) reduced the amount of NKCC1 accessible to basolateral surface biotinylation. Loss of cell surface NKCC1 was due to internalization as shown by 1) the resistance of biotinylated NKCC1 to surface biotin stripping after incubation with PMA and 2) indirect immunofluorescent labeling. PMA-induced internalization of NKCC1 is dependent on the -isoform of PKC as determined on the basis of sensitivity to a panel of PKC inhibitors. The effect of PMA on surface expression of NKCC1 was specific because PMA did not significantly alter the amount of Na+-K+-ATPase or E-cadherin available for surface biotinylation. After extended PMA exposure (>2 h), NKCC1 became degraded in a proteasome-dependent fashion. Like PMA, carbachol reduced the amount of NKCC1 accessible to basolateral surface biotinylation in a PKC--dependent manner. However, long-term exposure to carbachol did not result in degradation of NKCC1; rather, NKCC1 that was internalized after exposure to carbachol was recycled back to the cell membrane. PKC--dependent alteration of NKCC1 surface expression represents a novel mechanism for regulating Cl secretion. endocytosis; recycling; ion transporters 相似文献
12.
Harks EG Peters PH van Dongen JL van Zoelen EJ Theuvenet AP 《American journal of physiology. Cell physiology》2005,289(1):C130-C137
We have used normal rat kidney (NRK) fibroblasts as an in vitro model system to study cell transformation. These cells obtain a transformed phenotype upon stimulation with growth-modulating factors such as retinoic acid (RA) or transforming growth factor- (TGF-). Patch-clamp experiments showed that transformation is paralleled by a profound membrane depolarization from around 70 to 20 mV. This depolarization is caused by a compound in the medium conditioned by transformed NRK cells, which enhances intracellular Ca2+ levels and thereby activates Ca2+-dependent Cl channels. This compound was identified as prostaglandin F2 (PGF2) using electrospray ionization mass spectrometry. The active concentration in the medium conditioned by transformed NRK cells as determined using an enzyme immunoassay was 19.7 ± 2.5 nM (n = 6), compared with 1.5 ± 0.1 nM (n = 3) conditioned by nontransformed NRK cells. Externally added PGF2 was able to trigger NRK cells that had grown to density arrest to restart their proliferation. This proliferation was inhibited when the FP receptor (i.e., natural receptor for PGF2) was blocked by AL-8810. RA-induced phenotypic transformation of NRK cells was partially (25%) suppressed by AL-8810. Our results demonstrate that PGF2 acts as an autocrine enhancer and paracrine inducer of cell transformation and suggest that it may play a crucial role in carcinogenesis in general. membrane potential; intracellular calcium; mass spectrometry; FP receptor 相似文献
13.
Lyall P Armstrong WM Lyall V 《American journal of physiology. Cell physiology》2000,279(5):C1385-C1392
Effects of HCO3 on protein kinase C (PKC)-and protein kinase A (PKA)-induced anion conductances were investigatedin Necturus gallbladder epithelial cells. InHCO3-free media, activation of PKC via12-O-tetradecanoylphorbol 13-acetate (TPA) depolarizedapical membrane potential (Va) and decreased fractional apical voltage ratio (FR). These effects wereblocked by mucosal 5-nitro-2-(3-phenylpropylamino) benzoic acid(NPPB), a Cl channel blocker. In HCO3media, TPA induced significantly greater changes inVa and FR. These effects wereblocked only when NPPB was present in both mucosal and basolateralcompartments. The data suggest that TPA activates NPPB-sensitive apicalCl conductance (gCla) in theabsence of HCO3; in its presence, TPA stimulated bothNPPB-sensitive gCla and basolateralCl conductance (gClb).Activation of PKA via 3-isobutyl-1-methylxanthine (IBMX) also decreased Va and FR; however, thesechanges were not affected by external HCO3. Weconclude that HCO3 modulates the effects of PKC ongClb. In HCO3 medium, TPAand IBMX also induced an initial transient hyperpolarization andincrease in intracellular pH. Because these changes were independent ofmucosal Na+ and Cl, it is suggested that TPAand IBMX induce a transient increase in apical HCO3 conductance. 相似文献
14.
Mitochondrial Ca2+-induced K+ influx increases respiration and enhances ROS production while maintaining membrane potential 总被引:1,自引:0,他引:1
Heinen A Camara AK Aldakkak M Rhodes SS Riess ML Stowe DF 《American journal of physiology. Cell physiology》2007,292(1):C148-C156
We recently demonstrated a role for altered mitochondrial bioenergetics and reactive oxygen species (ROS) production in mitochondrial Ca2+-sensitive K+ (mtKCa) channel opening-induced preconditioning in isolated hearts. However, the underlying mitochondrial mechanism by which mtKCa channel opening causes ROS production to trigger preconditioning is unknown. We hypothesized that submaximal mitochondrial K+ influx causes ROS production as a result of enhanced electron flow at a fully charged membrane potential (m). To test this hypothesis, we measured effects of NS-1619, a putative mtKCa channel opener, and valinomycin, a K+ ionophore, on mitochondrial respiration, m, and ROS generation in guinea pig heart mitochondria. NS-1619 (30 µM) increased state 2 and 4 respiration by 5.2 ± 0.9 and 7.3 ± 0.9 nmol O2·min1·mg protein1, respectively, with the NADH-linked substrate pyruvate and by 7.5 ± 1.4 and 11.6 ± 2.9 nmol O2·min1·mg protein1, respectively, with the FADH2-linked substrate succinate (+ rotenone); these effects were abolished by the mtKCa channel blocker paxilline. m was not decreased by 1030 µM NS-1619 with either substrate, but H2O2 release was increased by 44.8% (65.9 ± 2.7% by 30 µM NS-1619 vs. 21.1 ± 3.8% for time controls) with succinate + rotenone. In contrast, NS-1619 did not increase H2O2 release with pyruvate. Similar results were found for lower concentrations of valinomycin. The increase in ROS production in succinate + rotenone-supported mitochondria resulted from a fully maintained m, despite increased respiration, a condition that is capable of allowing increased electron leak. We propose that mild matrix K+ influx during states 2 and 4 increases mitochondrial respiration while maintaining m; this allows singlet electron uptake by O2 and ROS generation. mitochondrial bioenergetics; heart mitochondria 相似文献
15.
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
16.
We have used the recombinantNH2-terminalmyc-tagged rabbitNa+-glucose transporter (SGLT1) tostudy the regulation of this carrier expressed in COS-7 cells.Treatment of cells with a protein kinase C (PKC) agonist, phorbol12-myristate 13-acetate (PMA), caused a significant decrease (38.03 ± 0.05%) in methyl-D-glucopyranoside transportactivity that could not be emulated by 4-phorbol 12,13-didecanoate. The decrease in sugar uptake stimulated by PMA was reversed by the PKCinhibitor bisindolylmaleimide I. The maximal rate ofNa+-glucose cotransport activity(Vmax) wasdecreased from 1.29 ± 0.09 to 0.85 ± 0.04 nmol · min1 · mgprotein1 after PMAexposure. However, measurement of high-affinityNa+-dependent phloridzin bindingrevealed that there was no difference in the number of cell surfacetransporters after PMA treatment; maximal binding capacities were 1.54 ± 0.34 and 1.64 ± 0.21 pmol/mg protein for untreated andtreated cells, respectively. The apparent sugar binding affinity(Michaelis-Menten constant) and phloridzin binding affinity(dissociation constant) were not affected by PMA. Because PKC reducedVmax withoutaffecting the number of cell surface SGLT1 transporters, we concludethat PKC has a direct effect on the carrier, resulting in a lowering ofthe transporter turnover rate by a factor of two. 相似文献
17.
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
18.
Maglova Lilia M.; Crowe William E.; Altamirano Anibal A.; Russell John M. 《American journal of physiology. Cell physiology》1998,275(2):C515
The effects ofhuman cytomegalovirus (HCMV) infection onCl/HCO3exchanger activity in human lung fibroblasts (MRC-5 cells) were studiedusing fluorescent, ion-sensitive dyes. The intracellular pH(pHi) of mock- and HCMV-infectedcells bathed in a solution containing 5%CO2-25 mMHCO3 were nearly the same. However,replacement of external Clwith gluconate caused anH2DIDS-inhibitable (100 µM)increase in the pHi ofHCMV-infected cells but not in mock-infected cells. Continuous exposureto hyperosmotic external media containing CO2/HCO3caused the pHi of both cell typesto increase. The pHi remainedelevated in mock-infected cells. However, in HCMV-infected cells, thepHi peaked and then recoveredtoward control values. This pHirecovery phase was completely blocked by 100 µMH2DIDS. In the presence ofCO2/HCO3, there was an H2DIDS-sensitivecomponent of net Cl efflux(external Cl wassubstituted with gluconate) that was less in mock- than in HCMV-infected cells. When nitrate was substituted for external Cl (in the nominal absenceofCO2/HCO3),the H2DIDS-sensitive netCl efflux was much greaterfrom HCMV- than from mock-infected cells. In mock-infected cells,H2DIDS-sensitive, netCl efflux decreased aspHi increased, whereas forHCMV-infected cells, efflux increased aspHi increased. All these resultsare consistent with an HCMV-induced enhancement ofCl/HCO3exchanger activity. 相似文献
19.
Francis H LeSage G DeMorrow S Alvaro D Ueno Y Venter J Glaser S Mancino MG Marucci L Benedetti A Alpini G 《American journal of physiology. Cell physiology》2007,293(4):C1252-C1262
Secretin stimulates ductal secretion by activation of cAMP PKA CFTR Cl–/HCO3– exchanger in cholangiocytes. We evaluated the expression of 2A-, 2B-, and 2C-adrenergic receptors in cholangiocytes and the effects of the selective 2-adrenergic agonist UK 14,304, on basal and secretin-stimulated ductal secretion. In normal rats, we evaluated the effect of UK 14,304 on bile and bicarbonate secretion. In bile duct-ligated (BDL) rats, we evaluated the effect of UK 14,304 on basal and secretin-stimulated 1) bile and bicarbonate secretion; 2) duct secretion in intrahepatic bile duct units (IBDU) in the absence or presence of 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na+/H+ exchanger isoform NHE3; and 3) cAMP levels, PKA activity, Cl– efflux, and Cl–/HCO3– exchanger activity in purified cholangiocytes. 2-Adrenergic receptors were expressed by all cholangiocytes in normal and BDL liver sections. UK 14,304 did not change bile and bicarbonate secretion of normal rats. In BDL rats, UK 14,304 inhibited secretin-stimulated 1) bile and bicarbonate secretion, 2) expansion of IBDU luminal spaces, and 3) cAMP levels, PKA activity, Cl– efflux, and Cl–/HCO3– exchanger activity in cholangiocytes. There was decreased lumen size after removal of secretin in IBDU pretreated with UK 14,304. In IBDU pretreated with EIPA, there was no significant decrease in luminal space after removal of secretin in either the absence or presence of UK 14,304. The inhibitory effect of UK 14,304 on ductal secretion is not mediated by the apical cholangiocyte NHE3. 2-Adrenergic receptors play a role in counterregulating enhanced ductal secretion associated with cholangiocyte proliferation in chronic cholestatic liver diseases. bicarbonate secretion; chloride efflux; gastrointestinal hormones; intrahepatic biliary epithelium; protein kinase A 相似文献
20.
Fan H Zingarelli B Peck OM Teti G Tempel GE Halushka PV Spicher K Boulay G Birnbaumer L Cook JA 《American journal of physiology. Cell physiology》2005,289(2):C293-C301
Heterotrimeric Gi proteins may play a role in lipopolysaccharide (LPS)-activated signaling through Toll-like receptor 4 (TLR4), leading to inflammatory mediator production. Although LPS is a TLR4 ligand, the gram-positive bacterium Staphylococcus aureus (SA) is a TLR2 ligand, and group B streptococci (GBS) are neither TLR2 nor TLR4 ligands but are MyD88 dependent. We hypothesized that genetic deletion of Gi proteins would alter mediator production induced by LPS and gram-positive bacterial stimulation. We examined genetic deletion of Gi2 or Gi1/3 protein in Gi2-knockout (Gi2/) or Gi1/3-knockout (Gi1/3/) mice. LPS-, heat-killed SA-, or GBS-induced mediator production in splenocytes or peritoneal macrophages (M) was investigated. There were significant increases in LPS-, SA-, and GBS-induced production of TNF- and IFN- in splenocytes from Gi2/ mice compared with wild-type (WT) mice. Also, LPS-induced TNF- was increased in splenocytes from Gi1/3/ mice. In contrast to splenocytes, LPS-, SA-, and GBS-induced TNF-, IL-10, and thromboxane B2 (TxB2) production was decreased in M harvested from Gi2/ mice. Also, LPS-induced production of IL-10 and TxB2 was decreased in M from Gi1/3/ mice. In subsequent in vivo studies, TNF- levels after LPS challenge were significantly greater in Gi2/ mice than in WT mice. Also, myeloperoxidase activity, a marker of tissue neutrophil infiltration, was significantly increased in the gut and lung of LPS-treated Gi2/ mice compared with WT mice. These data suggest that Gi proteins differentially regulate murine TLR-mediated inflammatory cytokine production in a cell-specific manner in response to both LPS and gram-positive microbial stimuli. Gi protein-deficient mice; endotoxin; group B streptococci; Staphylococcus aureus; Toll-like receptors 相似文献