共查询到20条相似文献,搜索用时 31 毫秒
1.
Wright DG Zhang Y Murphy JR 《Biochemical and biophysical research communications》2008,376(1):200-205
Peptide nucleic acid (PNA) is highly stable and binds to complementary RNA and DNA with high affinity, but it resists cellular uptake, thereby limiting its bioavailability. We investigated whether protectiveantigen (PA, a non-toxic component of anthrax toxin) could transport antisense PNA oligomers into reporter cells that contain luciferase transgenes with mutant β-globin IVS2 intronic inserts, which permit aberrant pre-mRNA splicing and impair luciferase expression. PNA oligomers antisense to mutant splice sites in these IVS2 inserts induced luciferase expression when effectively delivered into the cells. PNA 18-mers with C-terminal poly-lysine tails [PNA(Lys)8] demonstrated modest sequence-specific antisense activity by themselves at micromolar concentrations in luc-IVS2 reporter cell cultures. However, this activity was greatly amplified by PA. Antisense PNA(Lys)8 with but not without PA also corrected the IVS2-654 β-globin splice defect in cultured erythroid precursor cells from a patient with β-thalassemia [genotype, IVS2-654(β0/βE)], providing further evidence that anthrax PA can effectively transport antisense PNA oligomers into cells. 相似文献
2.
Da Silva N Shum WW El-Annan J Păunescu TG McKee M Smith PJ Brown D Breton S 《American journal of physiology. Cell physiology》2007,293(1):C199-C210
An acidic luminal pH in the epididymis contributes to maintaining sperm quiescent during their maturation and storage. The vacuolar H+ATPase (V-ATPase), located in narrow and clear cells, is a major contributor to luminal acidification. Mutations in one of the V-ATPase subunits, ATP6v1B1 (B1), cause distal renal tubular acidosis in humans but surprisingly, B1/ mice do not develop metabolic acidosis and are fertile. While B1 is located in the apical membrane of narrow and clear cells, the B2 subunit localizes to subapical vesicles in wild-type mouse, rat and human epididymis. However, a marked increase (84%) in the mean pixel intensity of B2 staining was observed in the apical pole of clear cells by conventional immunofluorescence, and relocalization into their apical membrane was detected by confocal microscopy in B1/ mice compared with B1+/+. Immunogold electron microscopy showed abundant B2 in the apical microvilli of clear cells in B1/ mice. B2 mRNA expression, determined by real time RT-PCR using laser-microdissected epithelial cells, was identical in both groups. Semiquantitative Western blots from whole epididymis and cauda epididymidis showed no variation of B2 expression. Finally, the luminal pH of the cauda epididymidis was the same in B1/ mice as in B1+/+ (pH 6.7). These data indicate that whereas overall expression of B2 is not affected in B1/ mice, significant redistribution of B2-containing complexes occurs from intracellular compartments into the apical membrane of clear cells in B1/ mice. This relocation compensates for the absence of functional B1 and maintains the luminal pH in an acidic range that is compatible with fertility. male reproductive tract; male fertility; luminal acidification; proton pump; vacuolar H+ATPase 相似文献
3.
Role of NBC1 in apical and basolateral HCO3- permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium 总被引:1,自引:0,他引:1
Corneal transparency and hydration control are dependent on HCO3 transport properties of the corneal endothelium. Recent work (13) suggested the presence of an apical 1Na+-3HCO3 cotransporter (NBC1) in addition to a basolateral 1Na+-2HCO3 cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO3 permeability and whether the cotransporter participates in transendothelial net HCO3 flux in cultured bovine corneal endothelium. NBC1 protein expression was reduced using small interfering RNA (siRNA). Immunoblot analysis showed that 515 nM siRNA decreased NBC1 expression by 8095%, 4 days posttransfection. Apical and basolateral HCO3 permeabilities were determined by measuring the rate of pHi change when HCO3 was removed from the bath under constant pH or constant CO2 conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO3 permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO3 permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO3 flux was 0.707 ± 0.009 mM·min1·cm2 in the basolateral-to-apical direction and increased to 1.74 ± 0.15 when cells were stimulated with 2 µM forskolin. Treatment with 5 nM siRNA decreased basolateral-to-apical flux by 67%, whereas apical-to-basolateral flux was unaffected, significantly decreasing net HCO3 flux to 0.236 ± 0.002. NBC1 siRNA treatment or 100 µM ouabain also eliminated steady-state HCO3 flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO3 permeability, basolateral-to-apical fluxes, and net HCO3 flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO3 flux and is functional only at the basolateral membrane. corneal endothelium; sodium bicarbonate cotransporter; small interfering RNA; bicarbonate transport 相似文献
4.
Ca2+ influx through alpha1S DHPR may play a role in regulating Ca2+ release from RyR1 in skeletal muscle 总被引:1,自引:0,他引:1
Shtifman A Paolini C López JR Allen PD Protasi F 《American journal of physiology. Cell physiology》2004,287(1):C73-C78
Skeletal muscle fiber types differ in their contents of total phosphate, which includes inorganic phosphate (Pi) and high-energy organic pools of ATP and phosphocreatine (PCr). At steady state, uptake of Pi into the cell must equal the rate of efflux, which is expected to be a function of intracellular Pi concentration. We measured 32P-labeled Pi uptake rates in different muscle fiber types to determine whether they are proportional to cellular Pi content. Pi uptake rates in isolated, perfused rat hindlimb muscles were linear over time and highest in soleus (2.42 ± 0.17 µmol·g1·h1), lower in red gastrocnemius (1.31 ± 0.11 µmol·g1·h1), and lowest in white gastrocnemius (0.49 ± 0.06 µmol·g1·h1). Reasonably similar rates were obtained in vivo. Pi uptake rates at plasma Pi concentrations of 0.31.7 mM confirm that the Pi uptake process is nearly saturated at normal plasma Pi levels. Pi uptake rate correlated with cellular Pi content (r = 0.99) but varied inversely with total phosphate content. Sodium-phosphate cotransporter (PiT-1) protein expression in soleus and red gastrocnemius were similar to each other and seven- to eightfold greater than PiT-1 expression in white gastrocnemius. That the PiT-1 expression pattern did not match the pattern of Pi uptake across fiber types implies that other factors are involved in regulating Pi uptake in skeletal muscle. Furthermore, fractional turnover of the cellular Pi pool (0.67, 0.57, and 0.33 h1 in soleus, red gastrocnemius, and white gastrocnemius, respectively) varies among fiber types, indicating differential management of intracellular Pi, likely due to differences in resistance to Pi efflux from the fiber. inorganic phosphate; sodium-inorganic phosphate transporters; PiT-2; inorganic phosphate efflux 相似文献
5.
Glutamine's protection against cellular injury is dependent on heat shock factor-1 总被引:12,自引:0,他引:12
Morrison AL Dinges M Singleton KD Odoms K Wong HR Wischmeyer PE 《American journal of physiology. Cell physiology》2006,290(6):C1625-C1632
Glutamine (GLN) has been shown to protect cells, tissues, and whole organisms from stress and injury. Enhanced expression of heat shock protein (HSP) has been hypothesized to be responsible for this protection. To date, there are no clear mechanistic data confirming this relationship. This study tested the hypothesis that GLN-mediated activation of the HSP pathway via heat shock factor-1 (HSF-1) is responsible for cellular protection. Wild-type HSF-1 (HSF-1+/+) and knockout (HSF-1/) mouse fibroblasts were used in all experiments. Cells were treated with GLN concentrations ranging from 0 to 16 mM and exposed to heat stress injury in a concurrent treatment model. Cell viability was assayed with phenazine methosulfate plus tetrazolium salt, HSP-70, HSP-25, and nuclear HSF-1 expression via Western blot analysis, and HSF-1/heat shock element (HSE) binding via EMSA. GLN significantly attenuated heat-stress induced cell death in HSF-1+/+ cells in a dose-dependent manner; however, the survival benefit of GLN was lost in HSF-1/ cells. GLN led to a dose-dependent increase in HSP-70 and HSP-25 expression after heat stress. No inducible HSP expression was observed in HSF-1/ cells. GLN increased unphosphorylated HSF-1 in the nucleus before heat stress. This was accompanied by a GLN-mediated increase in HSF-1/HSE binding and nuclear content of phosphorylated HSF-1 after heat stress. This is the first demonstration that GLN-mediated cellular protection after heat-stress injury is related to HSF-1 expression and cellular capacity to activate an HSP response. Furthermore, the mechanism of GLN-mediated protection against injury appears to involve an increase in nuclear HSF-1 content before stress and increased HSF-1 promoter binding and phosphorylation. knockout cells; amino acid; heat stress mechanism 相似文献
6.
Characterization of vectorial chloride transport pathways in the human pancreatic duct adenocarcinoma cell line HPAF 总被引:1,自引:0,他引:1
Fong P Argent BE Guggino WB Gray MA 《American journal of physiology. Cell physiology》2003,285(2):C433-C445
Pancreatic duct cells express a Ca2+-activated Cl- conductance (CaCC), upregulation of which may be beneficial to patients with cystic fibrosis. Here, we report that HPAF, a human pancreatic ductal adenocarcinoma cell line that expresses CaCC, develops into a high-resistance, anion-secreting epithelium. Mucosal ATP (50 µM) caused a fourfold increase in short-circuit current (Isc), a hyperpolarization of transepithelial potential difference (from -4.9 ± 0.73 to -8.5 ± 0.84 mV), and a fall in resistance to less than one-half of resting values. The effects of ATP were inhibited by mucosal niflumic acid (100 µM), implicating an apical CaCC in the response. RT-PCR indicated expression of hClC-2, hClC-3, and hClC-5, but surprisingly not hCLCA-1 or hCLCA-2. K+ channel activity was necessary to maintain the ATP-stimulated Isc. Using a pharmacological approach, we found evidence for two types of K+ channels in the mucosal and serosal membranes of HPAF cells, one activated by chlorzoxazone (500 µM) and sensitive to clotrimazole (30 µM), as well as one blocked by clofilium (100 µM) but not chromanol 293B (5 µM). RT-PCR indicated expression of the Ca2+-activated K+ channel KCNN4, as well as the acid-sensitive, four transmembrane domain, two pore K+ channel, KCNK5 (hTASK-2). Western blot analysis verified the expression of CLC channels, as well as KCNK5. We conclude that HPAF will be a useful model system for studying channels pertinent to anion secretion in human pancreatic duct cells. Ussing chamber; short-circuit current; RT-PCR; immunoblot 相似文献
7.
Li Z Zhou Z Saunders MM Donahue HJ 《American journal of physiology. Cell physiology》2006,290(4):C1248-C1255
Gap junctional channels between cells provide a pathway for exchange of regulatory ions and small molecules. We previously demonstrated that expression of connexins and cell-to-cell communication parallel osteoblastic differentiation and that nonspecific pharmacological inhibitors of gap junctional communication inhibit alkaline phosphatase activity. In this study, we stably transfected connexin (Cx)43 antisense cDNA into the immortalized human fetal osteoblastic cell line hFOB 1.19 (hFOB/Cx43). hFOB/Cx43 cells express lower levels of Cx43 protein and mRNA and display a 50% decrease in gap junctional intercellular communication relative to control [hFOB/plasmid vector control (pvc)]. This suggests that other connexins, such as Cx45, which is expressed to a similar degree in hFOB/Cx43 cells and hFOB/pvc cells, contribute to cell-to-cell communication in hFOB 1.19 cells. We observed almost total inhibition of alkaline phosphatase activity in hFOB/Cx43 cells despite only a 50% decrease in cell-to-cell communication. This suggests the intriguing possibility that Cx43 expression per se, independent of cell-to-cell communication, influences alkaline phosphatase activity and perhaps bone cell differentiation. Quantitative real-time RT-PCR revealed that mRNA levels for osteocalcin and core binding factor 1 (Cbfa1) increased as a function of time in hFOB/pvc but were inhibited in hFOB/Cx43. Osteopontin mRNA levels were increased in hFOB/Cx43 relative to hFOB/pvc and decreased as a function of time in both hFOB/Cx43 and hFOB/pvc. Transfection with Cx43 antisense did not affect expression of type I collagen in hFOB 1.19 cells. These results suggest that gap junctional intercellular communication and expression of Cx43 contribute to alkaline phosphatase activity, as well as osteocalcin, osteopontin, and Cbfa1 expression in osteoblastic cells. gap junction communication; alkaline phosphatase activity; osteopontin; osteocalcin; hFOB 1.19 相似文献
8.
Chicken thymocyte fractionation by peanut agglutinin lectin yields two cell subpopulations which differ in their GvH competence when injected into major histocompatibility complex (MHC)-(B locus) incompatible embryos. The nonagglutinated PNA? fraction displayed a degree of alloreactivity similar to that of peripheral blood lymphocytes while the PNA+ cells were nonreactive. Analysis of chickens at the age of 4 to 24 weeks showed that the development of GvH-reactive PBL preceded by 12 weeks the maturation of GvH-reactive PNA? thymocytes. Possible interpretations for this difference between the time of appearance of GvH reactivity of PNA? thymocytes and PBL are discussed. 相似文献
9.
Gagnon KB England R Delpire E 《American journal of physiology. Cell physiology》2006,290(1):C134-C142
In the present study, we have demonstrated functional interaction between Ste20-related proline-alanine-rich kinase (SPAK), WNK4 [with no lysine (K)], and the widely expressed Na+-K+-2Cl cotransporter type 1 (NKCC1). NKCC1 function, which we measured in Xenopus laevis oocytes under both isosmotic (basal) and hyperosmotic (stimulated) conditions, was unaffected when SPAK and WNK4 were expressed alone. In contrast, expression of both kinases with NKCC1 resulted in a significant increase in cotransporter activity and an insensitivity to external osmolarity or cell volume. NKCC1 activation is dependent on the catalytic activity of SPAK and likely also of WNK4, because mutations in their catalytic domains result in an absence of cotransporter stimulation. The results of our yeast two-hybrid experiments suggest that WNK4 does not interact directly with NKCC1 but does interact with SPAK. Functional experiments demonstrated that the binding of SPAK to WNK4 was also required because a SPAK-interaction-deficient WNK4 mutant (Phe997Ala) did not increase NKCC1 activity. We also have shown that the transport function of K+-Cl cotransporter type 2 (KCC2), a neuron-specific KCl cotransporter, was diminished by the expression of both kinases under both isosmotic and hyposmotic conditions. Our data are consistent with WNK4 interacting with SPAK, which in turn phosphorylates and activates NKCC1 and phosphorylates and deactivates KCC2. bumetanide; Na+-K+-2Cl cotransporter; K+-Cl cotransporter; Xenopus oocytes 相似文献
10.
Interactions of transmembrane carbonic anhydrase, CAIX, with bicarbonate transporters 总被引:2,自引:0,他引:2
Morgan PE Pastoreková S Stuart-Tilley AK Alper SL Casey JR 《American journal of physiology. Cell physiology》2007,293(2):C738-C748
Association of some plasma membrane bicarbonate transporters with carbonic anhydrase enzymes forms a bicarbonate transport metabolon to facilitate metabolic CO2-HCO3– conversions and coupled HCO3– transport. The transmembrane carbonic anhydrase, CAIX, with its extracellular catalytic site, is highly expressed in parietal and other cells of gastric mucosa, suggesting a role in acid secretion. We examined in transfected HEK293 cells the functional and physical interactions between CAIX and the parietal cell Cl–/HCO3– exchanger AE2 or the putative Cl–/HCO3– exchanger SLC26A7. Coexpression of CAIX increased AE2 transport activity by 28 ± 7% and also activated transport mediated by AE1 and AE3 (32 ± 10 and 37 ± 9%, respectively). In contrast, despite a transport rate comparable to that of AE3, coexpressed CAIX did not alter transport associated with SLC26A7. The CAIX-associated increase of AE2 activity did not result from altered AE2 expression or cell surface processing. CAIX was coimmunoprecipitated with the coexpressed SLC4 polypeptides AE1, AE2, and AE3, but not with SLC26A7. GST pull-down assays with a series of domain-deleted forms of CAIX revealed that the catalytic domain of CAIX mediated interaction with AE2. AE2 and CAIX colocalized in human gastric mucosa, as indicated by coimmunofluorescence. This is the first example of a functional and physical interaction between a bicarbonate transporter and a transmembrane carbonic anhydrase. We conclude that CAIX can bind to some Cl–/HCO3– exchangers to form a bicarbonate transport metabolon. SLC4; SLC26; bicarbonate transport metabolon 相似文献
11.
Nejsum LN Praetorius J Nielsen S 《American journal of physiology. Cell physiology》2005,289(2):C333-C340
In isolated sweat glands, bumetanide inhibits sweat secretion. The mRNA encoding bumetanide-sensitive Na+-K+-Cl cotransporter (NKCC) isoform 1 (NKCC1) has been detected in sweat glands; however, the cellular and subcellular protein localization is unknown. Na+/H+ exchanger (NHE) isoform 1 (NHE1) protein has been localized to both the duct and secretory coil of human sweat duct; however, the NHE1 abundance in the duct was not compared with that in the secretory coil. The aim of this study was to test whether mRNA encoding NKCC1, NKCC2, and Na+-coupled acid-base transporters and the corresponding proteins are expressed in rodent sweat glands and, if expressed, to determine the cellular and subcellular localization in rat, mouse, and human eccrine sweat glands. NKCC1 mRNA was demonstrated in rat palmar tissue, including sweat glands, using RT-PCR, whereas NKCC2 mRNA was absent. Also, NHE1 mRNA was demonstrated in rat palmar tissue, whereas NHE2, NHE3, NHE4, electrogenic Na+-HCO3 cotransporter 1 NBCe1, NBCe2, electroneutral Na+-HCO3 cotransporter NBCn1, and Na+-dependent Cl/HCO3 exchanger NCBE mRNA were not detected. The expression of NKCC1 and NHE1 proteins was confirmed in rat palmar skin by immunoblotting, whereas NKCC2, NHE2, and NHE3 proteins were not detected. Immunohistochemistry was performed using sections from rat, mouse, and human palmar tissue. Immunoperoxidase labeling revealed abundant expression of NKCC1 and NHE1 in the basolateral domain of secretory coils of rat, mouse, and human sweat glands and low expression was found in the coiled part of the ducts. In contrast, NKCC1 and NHE1 labeling was absent from rat, mouse, and human epidermis. Immunoelectron microscopy demonstrated abundant NKCC1 and NHE1 labeling of the basolateral plasma membrane of mouse sweat glands, with no labeling of the apical plasma membranes or intracellular structures. The basolateral NKCC1 of the secretory coils of sweat glands would most likely account for the observed bumetanide-sensitive NaCl secretion in the secretory coils, and the basolateral NHE1 is likely to be involved in Na+-coupled acid-base transport. bumetanide; eccrine glands; immunohistochemistry; immunoblotting 相似文献
12.
Following 20 d of exposure to 75 or 150 mol m3 NaCl Sorghumbicolor (L.) Moench plants become capable of growing in mediumcontaining 300 mol m3 NaCl. Control plants, which havenot been pretreated, or plants pretreated for less than 20 ddie within 2 weeks when exposed to 300 mol m3 NaCl. Weconsider this induction of a capacity to survive in and toleratea high NaCl concentration as an adaptation to salinity. We suggestthat adaptation to salinity is more than osmotic adjustmentand that it takes longer to develop than osmotic adjustment.Concomitantly with the appearance of the ability to grow inhigh salinity, adaptation also comprises the development ofa capacity to regulate internal Na+ and Cl concentrations,even when external salinity is high. Shoot mean relative growthrates are similar for both control plants and for adapted plantsgrowing in 300 mol m3 NaCl, although their shoot Na+and Cl concentrations are quite different. Based on thesedata, we propose that adaptation of Sorghum to high salinityresults from a modulation of genome expression occurring duringextended exposure to non-lethal NaCl concentrations. Key words: Sorghum bicolor (L.) Moench, NaCl, salt tolerance, adaptation to salinity 相似文献
13.
Huang D Khoe M Befekadu M Chung S Takata Y Ilic D Bryer-Ash M 《American journal of physiology. Cell physiology》2007,292(4):C1339-C1352
Focal adhesion kinase (FAK) is important to cellular functions such as proliferation, migration, and survival of anchorage-dependent cells. We investigated the role of FAK in modulating normal cellular responses, specifically cell survival in response to inflammatory stimuli and serum withdrawal, using FAK-knockout (FAK/) embryonic fibroblasts. FAK/ fibroblasts were more vulnerable to TNF--induced apoptosis, as measured by terminal deoxynucleotidyl transferase positivity. FAK/ fibroblasts also demonstrated increased procaspase-3 cleavage to p17 subunit, whereas this was undetectable in FAK+/+ fibroblasts. Insulin receptor substrate-1 expression was completely abolished and NF-B activity was reduced, with a concomitant decrease in abundance of the anti-apoptotic protein Bcl-xL in FAK/ cells. Upon serum withdrawal, FAK+/+ cells exhibited marked attenuation of basal ERK phosphorylation, while FAK/ cells, in contrast, maintained high basal ERK phosphorylation. Moreover, inhibition of ERK phosphorylation potentiated serum withdrawal-induced caspase-3 activity. This was paralleled by increased insulin receptor substrate (IRS)-2 expression in FAK/ cells, although both insulin- and IGF-1-mediated phosphorylation of Akt/PKB and GSK-3 were impaired. This suggests that IRS-2 protects against apoptosis upon serum withdrawal via the ERK signaling pathway. The specific role of FAK to protect cells from apoptosis is regulated by activation and phosphorylation of NF-B and interaction between activated growth factor anti-apoptotic signaling pathways involving both phosphatidylinositol 3-kinase/Akt and MAPK/ERK1/2. We demonstrate that FAK is necessary for upregulation of the anti-apoptotic NF-B response, as well as for normal expression of growth factor signaling proteins. Thus we propose a novel role for FAK in protection from cytokine-mediated apoptosis. apoptosis; ERK1/2; insulin; TNF-; IGF-1 相似文献
14.
Lepic E Burger D Lu X Song W Feng Q 《American journal of physiology. Cell physiology》2006,291(6):C1240-C1246
We recently demonstrated that deficiency in endothelial nitric oxide synthase (eNOS) results in congenital septal defects and postnatal heart failure. The aim of this study was to investigate the role of eNOS in cardiomyocyte proliferation and maturation during postnatal development. Cultured eNOS knockout (eNOS/) cardiomyocytes displayed fewer cells and lower bromodeoxyuridine (BrdU) incorporation in vitro compared with wild-type (WT) cardiomyocytes (P < 0.05). Treatment with the nitric oxide (NO) donor diethylenetriamine NONOate increased BrdU incorporation and cell counts in eNOS/ cardiomyocytes (P < 0.05). Inhibition of nitric oxide synthase activity using NG-nitro-L-arginine methyl ester decreased the level of BrdU incorporation and cell counts in WT cardiomyocytes (P < 0.05). Vascular endothelial growth factor (VEGF) increased the level of BrdU incorporation in cultured WT cardiomyocytes in a dose- and time-dependent manner (P < 0.05). Conversely, VEGF did not alter BrdU incorporation in eNOS/ cardiomyocytes (P = not significant). Furthermore, deficiency in eNOS significantly decreased BrdU labeling indexes in neonatal hearts in vivo. Although WT hearts displayed a rapid decrease in atrial natriuretic peptide (ANP) expression in the first week of neonatal life, ANP expression in eNOS/ hearts remain elevated. Our study demonstrated that NO production from eNOS is necessary for postnatal cardiomyocyte proliferation and maturation, suggesting that eNOS plays an important role during postnatal heart development. proliferation; heart development 相似文献
15.
Hershman Kenneth M.; Levitan Edwin S. 《American journal of physiology. Cell physiology》2000,278(2):C397
Previously, we reported that cell-cell contact regulatesK+ channel mRNA expression in cultured adult rat cardiacmyocytes. Here we show that exposing cardiac myocytes to tyrosinekinase inhibitors (genistein, tyrphostin A25), but not inactiveanalogs, prevents downregulation of Kv1.5 mRNA and upregulation ofKv4.2 mRNA normally observed when they are cultured under low-density conditions. Furthermore, cardiac myocytes cocultured with cells thatendogenously (Mv 1 Lu) or heterologously (Chinese hamster ovary cells)express the receptor-type protein tyrosine phosphatase µ (RPTPµ)display Kv1.5 mRNA levels paralleling that which was observed inmyocytes cultured under high-density conditions and in intact tissue.In contrast, myocytes cocultured with control cells failed to producethis response. Finally, it is shown that Kv4.2 mRNA expression isunaffected by RPTPµ. These findings reveal that multiple tyrosinephosphorylation-dependent mechanisms control cardiac myocyteK+ channel genes. Furthermore, we conclude that RPTPµspecifically regulates cardiac myocyte Kv1.5 mRNA expression. Thus thisreceptor protein tyrosine phosphatase may be important in responses to pathological conditions associated with the loss of cell-cell interactions in the heart. 相似文献
16.
Lewandowski ED O'donnell JM Scholz TD Sorokina N Buttrick PM 《American journal of physiology. Cell physiology》2007,292(5):C1880-C1886
Glucose metabolism in the heart requires oxidation of cytosolic NADH from glycolysis. This study examines shuttling reducing equivalents from the cytosol to the mitochondria via the activity and expression of the oxoglutarate-malate carrier (OMC) in rat hearts subjected to 2 wk (Hyp2, n = 6) and 10 wk (Hyp10, n = 8) of pressure overload hypertrophy vs. that of sham-operated rats (Sham2, n = 6; and Sham10, n = 7). Moderate aortic banding produced increased atrial natriuretic factor (ANF) mRNA expression at 2 and 10 wk, but only at 10 wk did hearts develop compensatory hypertrophy (33% increase, P < 0.05). Isolated hearts were perfused with the short-chain fatty acid [2,4-13C2]butyrate (2 mM) and glucose (5 mM) to enable dynamic-mode 13C NMR of intermediate exchange across OMC. OMC flux increased before the development of hypertrophy: Hyp2 = 9.6 ± 2.1 vs. Sham2 = 3.7 ± 1.2 µM·min1·g dry wt1, providing an increased contribution of cytosolic NADH to energy synthesis in the mitochondria. With compensatory hypertrophy, OMC flux returned to normal: Hyp10 = 3.9 ± 1.7 vs. Sham10 = 3.8 ± 1.2 µM·g1·min1. Despite changes in activity, no differences in OMC expression occurred between Hyp and Sham groups. Elevated OMC flux represented augmented cytosolic NADH shuttling, coupled to increased nonoxidative glycolysis, in response to hypertrophic stimulus. However, development of compensatory hypertrophy moderated the pressure-induced elevation in OMC flux, which returned to control levels. The findings indicate that the challenge of pressure overload increases cytosolic redox state and its contribution to mitochondrial oxidation but that hypertrophy, before decompensation, alleviates this stress response. malate-aspartate shuttle; redox state; hypertrophy 相似文献
17.
Critical role of p63 in the development of a normal esophageal and tracheobronchial epithelium 总被引:4,自引:0,他引:4
Daniely Y Liao G Dixon D Linnoila RI Lori A Randell SH Oren M Jetten AM 《American journal of physiology. Cell physiology》2004,287(1):C171-C181
The trachea and esophagus originate from the foregut endoderm during early embryonic development. Their epithelia undergo a series of changes involving the differentiation of stem cells into unique cell types and ultimately forming the mature epithelia. In this study, we monitored the expression of p63 in the esophagus and the trachea during development and examined in detail morphogenesis in p63/ mice. At embryonic day 15.5 (E15.5), the esophageal and tracheobronchial epithelia contain two to three layers of cells; however, only the progenitor cells express p63. These progenitor cells differentiate first into ciliated cells (p63/-tubulin IV+) and after birth into mature basal cells (p63+/K14+/K5+/BS-I-B4+). In the adult pseudostratified, columnar tracheal epithelium, K14+/K5+/BS-I-B4+ basal cells stain most intensely for p63, whereas ciliated and mucosecretory cells are negative. In stratified squamous esophageal epithelium and during squamous metaplasia in the trachea, cells in the basal layer stain strongest for p63, whereas p63 staining declines progressively in transient amplifying and squamous differentiated cells. Generally, p63 expression is restricted to human squamous cell carcinomas, and adenocarcinomas and Barrett's metaplasia do not stain for p63. Examination of morphogenesis in newborn p63/ mice showed an abnormal persistence of ciliated cells in the esophagus. Significantly, in both tissues, lack of p63 expression results in the development of a highly ordered, columnar ciliated epithelium deficient in basal cells. These observations indicate that p63 plays a critical role in the development of normal esophageal and tracheobronchial epithelia and appears to control the commitment of early stem cells into basal cell progeny and the maintenance of basal cells. retinoic acid; stem cell; carcinoma; basal cell; differentiation 相似文献
18.
Li W Nakagawa T Koyama N Wang X Jin J Mizuno-Horikawa Y Gu J Miyoshi E Kato I Honke K Taniguchi N Kondo A 《Glycobiology》2006,16(10):1007-1019
Alpha1,6-fucosyltransferase (Fut8) plays important roles inphysiological and pathological conditions. Fut8-deficient (Fut8/)mice exhibit growth retardation, earlier postnatal death, andemphysema-like phenotype. To investigate the underlying molecularmechanism by which growth retardation occurs, we examined themRNA expression levels of Fut8/ embryos (18.5days postcoitum [dpc]) using a cDNA microarray. The DNA microarrayand real-time polymerase chain reaction (PCR) analysis showedthat a group of genes, including trypsinogens 4, 7, 8, 11, 16,and 20, were down-regulated in Fut8/ embryos.Consistently, the expression of trypsinogen proteins was foundto be lower in Fut8/ mice in the duodenum, smallintestine, and pancreas. Trypsin, an active form of trypsinogen,regulates cell growth through a G-protein-coupled receptor,the proteinase-activated receptor 2 (PAR-2). In a cell culturesystem, a Fut8 knockdown mouse pancreatic acinar cell carcinoma,TGP49-Fut8-KDs, showed decreased growth rate, similar to thatseen in Fut8/ mice, and the decreased growth ratewas rescued by the application of the PAR-2-activating peptide(SLIGRL-NH2). Moreover, epidermal growth factor (EGF)-inducedreceptor phosphorylation was attenuated in TGP49-Fut8-KDs, whichwas highly associated with a reduction of trypsinogens mRNAlevels. The addition of exogenous EGF recovered c-fos, c-jun,and trypsinogen mRNA expression in TGP49-Fut8-KDs. Again, theEGF-induced up-regulation of c-fos and c-jun mRNA expressionwas significantly blocked by the protein kinase C (PKC) inhibitor.Our findings clearly demonstrate a relationship between Fut8and the regulation of EGF receptor (EGFR)-trypsin-PAR-2 pathwayin controlling cell growth and that the EGFR-trypsin-PAR-2 pathwayis suppressed in TGP49-Fut8-KDs as well as in Fut8/mice. 相似文献
19.
The Cl fluxes across the plasma membrane and the Clcontent of the acidresistant alga Dunaliella acidophila(optimal growthat pH 1.0, positive membrane potential) werestudied in the presence of 0.01300 mM Cl. Up to40 mM Cl in the medium, theinternal Cl concentrationis higher than that predicted by the electrochemical equilibrium,whereas at higher external Cl concentrations internalCl levels are lower than expected for the electrochemicalequilibrium. Growth in the absence of Cl is significantlylower than in the standard growth medium (2.2 mM Cl)and this reduction cannot be overcome by the addition ofothermonovalent anions such as Br or NO3 The latterimplies a specific Cl requirement in addition to therole of Cl as apermeant anion during ion translocations.Growth and photosynthesis tolerate an excess of Cl upto 300 mM (without stepwiseadaptation to increasing salinity).The uptake of Cl (measured by tracer techniques) exhibitsMichaelisMenten kinetics (KM = 0.75 mM Cl) andis stimulated by light and high H+ concentrations. Internalacidification by acetic acid causes an inhibition of Cluptake. The uptake of Cl is inhibited by the monovalentanions Br, I, and NO3 with K1, values notvery much different from the KM. value for Cl. The aniontransport inhibitors SITS and DIDS do not affect photosynthesis,but strongly suppressthe uptake of Cl. The Clchannel blockers A9C and NPPB cause inhibitionsof Cl uptake as well as of photosynthesis andthe ATPpool. FCCP strongly depresses the internal ATPpool withouta marked effect on Cl uptake. Cl efflux was inhibitedbyDIDS and SITS, but stimulated or inhibited by FCCP, dependingon the external Cl concentration. Results are in agreementwiththe hypothesis that Cl uptake into D. acidophila is dueto catalysed diffusion and is primarily independent of the hydrolysisofATP. Cl efflux is assumed to be coupled to an activepump. Data suggest tight cooperativity between the systemsresponsiblefor Cl uptake and Cl efflux, with thecytoplasmic pH and the membrane potential being important mediators. Key words: Acid resistance, chloride carrier, chloride channels, Dunaliella acidophila, membrane potential, plasma membrane 相似文献
20.
Yeung HM Kravtsov GM Ng KM Wong TM Fung ML 《American journal of physiology. Cell physiology》2007,292(6):C2046-C2056
This study examined Ca2+ handling mechanisms involved in cardioprotection induced by chronic intermittent hypoxia (CIH) against ischemia-reperfusion (I/R) injury. Adult male Sprague-Dawley rats were exposed to 10% inspired O2 continuously for 6 h daily from 3, 7, and 14 days. In isolated perfused hearts subjected to I/R, CIH-induced cardioprotection was most significant in the 7-day group with less infarct size and lactate dehydrogenase release, compared with the normoxic group. The I/R-induced alterations in diastolic Ca2+ level, amplitude, time-to-peak, and the decay time of both electrically and caffeine-induced Ca2+ transients measured by spectrofluorometry in isolated ventricular myocytes of the 7-day CIH group were less than that of the normoxic group, suggesting an involvement of altered Ca2+ handling of the sarcoplasmic reticulum (SR) and sarcolemma. We further determined the protein expression and activity of 45Ca2+ flux of SR-Ca2+-ATPase, ryanodine receptor (RyR) and sarcolemmal Na+/Ca2+ exchange (NCX) in ventricular myocytes from the CIH and normoxic groups before and during I/R. There were no changes in expression levels of the Ca2+-handling proteins but significant increases in the RyR and NCX activities were remarkable during I/R in the CIH but not the normoxic group. The augmented RyR and NCX activities were abolished, respectively, by PKA inhibitor (0.5 µM KT5720 or 0.5 µM PKI14-22) and PKC inhibitor (5 µM chelerythrine chloride or 0.2 µM calphostin C) but not by Ca2+/calmodulin-dependent protein kinase II inhibitor KN-93 (1 µM). Thus, CIH confers cardioprotection against I/R injury in rat cardiomyocytes by altered Ca2+ handling with augmented RyR and NCX activities via protein kinase activation. cardioprotection; intracellular calcium 相似文献