共查询到20条相似文献,搜索用时 31 毫秒
1.
The number and activity of ouabain-sensitive Na/K ATPase pumps expressed by many cell types in vitro, including human retinal pigment epithelial cells (RPE), have been shown to decline with increasing culture density. Cell proliferation also declined as cultures became dense so it was unclear if pump number was modulated by cell proliferation or culture confluency. By exposing RPE cultures to various feeding regimens, using culture medium containing or lacking serum, it was possible to produce RPE cultures with a range of culture densities and growth rates. These were analyzed for proliferative activity by quantifying [3H]thymidine incorporation and for Na/K ATPase pump number by measuring specific [3H]ouabain binding. The results suggest that pump number is modulated by culture density and, further, that the density-dependent regulation of pump number requires serum. Although density-dependent modulation of culture growth is also serum requiring, cell proliferation and pump number did not appear to be related; cultures of similar density which differed significantly in growth rate had similar numbers of pumps. The view that elevated numbers of pumps were not necessarily found in proliferating cells was further supported by qualitative examination of radioautographs of cells dually labeled with [3H]thymidine and [3H]ouabain. Cycling cells which had [3H]thymidine-labeled nuclei did not have notably higher labeling with [3H]ouabain. However, [3H]ouabain labeling, as an indicator of pump site number and distribution, did vary among cells in an RPE population and also within individual cells. This latter observation suggests that unpolarized RPE cells in sparse cultures may have regionally different requirements for ionic regulation. 相似文献
2.
A study was made of the dependence of ATP hydrolysis intensity upon different ratios of sodium and potassium ions in plasma membrane of L cells and of cells of clone Lebr 625, sensitive and resistant to ethidium bromide, and of the distribution of cells according to cell cycle phases in dense and sparse cultures. In dense cultures, the cell growth is arrested on G1 phase, the hydrolytic activity of (Na+ + K+)-ATPase decreases, and the Na+, K+ ratio for maximum activity of (Na+ + K+)-ATPase changes. The higher proliferative activity of Lebr 625 cells in dense culture corresponds to the higher hydrolytic activity of (Na+ + K+)-ATPase. 相似文献
3.
Reversal by EGTA of the enhanced secretory responsiveness of mast cells due to treatment with ouabain 总被引:1,自引:0,他引:1
The effect of EGTA on the enhancement by ouabain of compound 48/80-induced secretion from mast cells was compared with the effect on the Na(+)-K+ pump activity. The time-dependent secretory enhancement by ouabain was blocked by addition of EGTA to the cell suspension concomitantly with the addition of ouabain, and EGTA caused a large increase in the pump activity. Addition of 10 microM EGTA to ouabain-treated cells stopped but did not reverse the enhancement. The experiments show that the effect of ouabain was due to changes in a calcium pool utilized in compound 48/80-induced secretion following changes in the Na+,K+ pump activity. 相似文献
4.
Activities related to Na-K transport were measured in cell cultures of ground squirrel kidney cortex in order to compare these cells with those of intact kidney and of continuous cell lines. A microsomal preparation containing plasma membrane Na,K-ATPase from fresh kidney showed twice the activity of a similar preparation from 72-hour cultured cells. Na,K-ATPase of homogenates of 72-hour cells showed one-third to one-fourth the specific activity of that from 6-hour cultured cells. The associated K-dependent phosphatase activity also declined as a function of time in culture. The ouabain-sensitive influx of K into 6-hour cultured cells was twice as great as the K influx into 72-hour cells. The number of sites binding 3H-ouabain in intact cultured cells declined 81% on a cell protein basis between 6 and 72 hours in culture. This decline in ouabain binding sites was relatively greater than that of K influx, so that the K turnover number increased over this same time period. The decline in ouabain-sensitive K influx during culture was complementary to an increase in furosemide-sensitive K influx. Measurements of unidirectional and net K fluxes showed that there were three components of K influx into 3-day cultured cells: ouabain-sensitive Na:K exchange, furosemide-sensitive K:K exchange, and K diffusion. In the 6-hour cultures, however, there was no furosemide-sensitive K:K exchange. Thus, after three days in culture ground squirrel kidney cells lose a feature characteristic of the original parent cells (high Na,K-ATPase activity), and gain a feature common to many undifferentiated cultured cells (furosemide-sensitive K:K exchange). 相似文献
5.
Cellular distribution and biological activity of epidermal growth factor receptors in A431 cells are influenced by cell-cell contact 总被引:4,自引:0,他引:4
The potential significance of cell-cell interactions on EGF receptor (EGFR) activity was investigated in cultured adherent A431 cells seeded as single-cell suspensions with different initial cell densities. In dense cultures, EGFRs were mainly localised at cell boundaries and in microvilli as shown by immunofluorescence analysis with an EGFR-specific antibody while in sparse cultures the distribution of EGFRs was more diffuse. Scatchard analysis showed that as cell density decreased the number of high-affinity receptors increased considerably. Upon treatment of adherent intact cells with EGF all cells in sparse cultures contained activated EGFRs as demonstrated by immunofluorescence analysis with a phosphotyrosine-specific antibody, while in dense cultures mainly cells at the periphery of a cluster and especially at their expanding borders exhibited activated EGFRs. EGF-induced phosphorylation in intact cells was greatly enhanced in sparse compared with dense cultures as demonstrated by immunoprecipitation with a phosphotyrosine-specific antibody. In contrast to intact cells, in cytoskeleton preparations, obtained after mild detergent treatment of adherent cells, EGFRs were able to undergo EGF-independent phosphorylation. Pretreatment of cells with EGF led to enhanced tyrosine phosphorylation of cytoskeletal-associated proteins. Our observations suggest that cell density has a considerable effect on the subcellular localisation as well as biological activity of the EGFR. Thus, in intact A431 cells growing with extensive cell-cell interactions some negative control mechanisms preventing EGFR activation may be exerted by adjacent cells. 相似文献
6.
K B Aslanidi L M Boitsova LJuChailakhyan L N Kublik I I Marachova T V Potapova T A Vinogradova 《FEBS letters》1991,283(2):295-297
Low ouabain concentration (1 x 10(-6) M) is shown to decrease intracellular K+ (K+in) and to increase intracellular Na+ (Na+in) in human fibroblast cell cultures. The same ouabain concentration was without effect upon K+in ad Na+in in rodent cultures such as BHK-21, mouse fibroblasts and rat glyoma C6 cells. K+in and Na+in in the mixed cultures of human and BHK-21 fibroblasts or human and mouse fibroblasts were found to be resistant to 1 x 10(-6) M ouabain whereas that of the mixtures of human and rat glyoma C6 cells proved to be ouabain-sensitive. The gap-junction-mediated dye transfer was revealed between human and BHK-21 cells. Such an effect was very small in the human-C6 cell mixed culture. It is concluded that cells with active ion pumps can support the maintenance of K+ and Na+ gradients in cells with inactive pumps, provided that effective ion transport via gap junctions takes place. 相似文献
7.
Effects of cell density and extracellular matrix on the lateral diffusion of major histocompatibility antigens in cultured fibroblasts 总被引:7,自引:5,他引:2
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
《The Journal of cell biology》1986,103(1):215-222
We have studied the effect of cell density on the lateral diffusion of major histocompatibility (MHC) antigens in the plasma membranes of fibroblasts using fluorescence recovery after photobleaching. The percent recovery of fluorescence was decreased in fibroblasts grown in confluent cultures. While recovery of fluorescence was measurable in greater than 90% of the cells from sparse cultures, measurable recovery was detected in only 60-80% of the cells from dense cultures; no mobile antigens were detectable in 20-40% of cells examined. The diffusion coefficient on human skin fibroblast cells that did show recovery was the same for cells grown in sparse or dense conditions. In WI-38, VA-2, and c1 1d cultures the diffusion coefficients of mobile antigens were smaller in cells from dense cultures. Changes in lateral diffusion occurred with increased cell-cell contact and with age of cell culture but were not observed in growth-arrested cells or in sparse cells cultured in medium conditioned by confluent cells. Decreased mobile fractions of MHC antigens were observed when cells were plated on extracellular matrix materials derived from confluent cultures. Treatment of the extracellular matrix materials with a combination of proteolytic enzymes or by enzymes that degrade proteoglycans abolished this effect. Matrices produced by cells from other cell lines were less effective in inducing changes in mobile fractions and purified matrix components alone did not induce changes in lateral diffusion. Finally, there were no differences in the proportion of MHC antigens that were resistant to Triton X-100 extraction in sparse and dense cells. These results suggest that cell-cell interactions mediated through extracellular matrix materials can influence the lateral diffusion of at least part of the population of MHC antigens. 相似文献
8.
ATP plus Mg2+ plus Na+ supported [3H]ouabain binding to canine left ventricular tissue homogenates and microsomal (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity from the same tissue were measured. A linear relationship was found between the initial velocity of [3H]ouabain binding to tissue homogenates and microsomal (Na+ + K+)-ATPase activity from the same tissue in the presence and absence of in vivo bound digoxin. In vivo bound digoxin reduced both measurements. With tissue from digoxin-free hearts, a linear relationship was also obtained between the initial velocity and the maximum level of [3H]ouabain binding to tissue homogenate. Binding of [3H]ouabain to whole tissue homogenate is a convenient method for estimating (Na+ + K+)-ATPase activity in small left ventricular biopsy samples. 相似文献
9.
R Moretti M Martín T Proverbio F Proverbio R Marín 《Comparative biochemistry and physiology. B, Comparative biochemistry》1991,98(4):623-626
1. Two Na(+)-stimulated ATPase activities were determined in gill homogenates from squid, shrimp and teleost fish; in kidney slice homogenates from teleost fish, bullfrog, toad, iguana, chicken, duck, rat, pig and cow, as well as in homogenates from rat small intestinal cells, brain cortex and liver slices. The two Na(+)-stimulated ATPase activities, the Na- and the Na,K-ATPase, showed a different behavior toward K+ and ouabain. 2. The ouabain-insensitive, K(+)-independent, Na-ATPase activity for all the studied homogenates was completely inhibited by 2 mM furosemide. 3. An increase in cell volume of the kidney, brain cortex and liver slice preparations, as well as of the rat small intestinal cells, produced a concomitant increase of the ouabain-insensitive Na-ATPase. 相似文献
10.
Brodsky VY Zvezdina ND Nechaeva NV Avdonin PV Novikova TE Gvasava IG Fateeva VI Malchenko LA 《Cell biology international》2003,27(12):965-976
Ultradian protein synthesis rhythm was used as a marker of cell cooperation in synchronous dense and non-synchronous sparse hepatocyte cultures. Phenylephrine (2 microM, 2 min), an alpha (1)-adrenoreceptor agonist, which exerts [Ca(2+)](cyt)elevation from intracellular stores, affected protein synthesis rhythm in sparse cultures, i.e. initiated cooperative activity of the cells. The same effect was produced by 2,5-di(tertiary-butyl)-1,4-benzohydroquinone (10 microM, 2 min), which increases [Ca(2+)](cyt)by a non-receptor pathway. Pretreatment of dense cultures with the intracellular calcium chelator, 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'- tetraacetic acid (acetoxymethyl) ester (BAPTA-AM) at 10-20 microM for, 30-60 min resulted in loss of the rhythm of protein synthesis, i.e. loss of cooperative activity between the cells. The medium conditioned by control dense cultures initiated rhythm in sparse cultures, whereas the conditioned medium of cultures pretreated with BAPTA-AM did not. [Ca(2+)](cyt)increase is known to occur with monosialoganglioside GM1 treatment. By ELISA estimation, the GM1 content in 3 h conditioned medium was similar in control dense cultures to that in cultures pretreated with BAPTA-AM. Bearing in mind data on the Ca(2+)-dependence of vesicle formation and shedding, the conditioned medium was separated by 150000 g centrifugation to supernatant containing monomers and micelles, and a pellet containing vesicular form of gangliosides. Only the latter initiated cooperative activity of the cells of sparse cultures. These cultures were also synchronized by GM1-containing liposomes at lower concentrations than added free GM1, 0.0003 and 0.06 microM respectively. Thus, GM1 and calcium are both involved in cell-cell synchronization. Activation of gangliosides, including GM1 and elevation of [Ca(2+)](cyt,)is known to lead to changes of protein kinase activity and protein phosphorylation resulting in modulation of oscillations in protein metabolism. 相似文献
11.
Modulation of functional and optimal (Na+-K+)ATPase activity during the cell cycle of neuroblastoma cells 总被引:2,自引:0,他引:2
Functional and optimal activities of the (Na+-K+)ATPase, as determined by ouabain-sensitive K+ influx in intact cells and ATP hydrolysis in cell homogenates respectively, have been measured during the cell cycle of neuroblastoma (clone Neuro-2A) cells. The cells were synchronized by selective detachment of mitotic cells. The ouabain-sensitive K+ influx decreased more than fourfold from 1.62 +/- 0.11 nmoles/min/10(6) cells to 0.36 +/- 0.25 nmoles/min/10(6) cells on passing from mitosis to early G1 phase. On entry into S phase a transient sixfold increase to 2.07 +/- 0.30 nmoles/min/10(6) cells was observed, followed by a rapid decline, after which the active K+ influx rose again steadily from 1.03 +/- 0.25 nmoles/min/10(6) cells in early S phase to 2.10 +/- 0.92 nmoles/min/10(6) cells just prior to the next mitosis. The ouabain-insensitive component rose linearly through the cycle in the same manner as the protein content/cell. Combining total K+ influx values with efflux data obtained previously showed that net loss of K+ occurred with transition from mitosis to G1 phase while net accumulation occurred with entry into S. Throughout mid-S phase net K+ flux was virtually zero, but a large net influx occurred again just before the next mitosis. The (Na+-K+)ATPase activity measured in cell homogenates decreased rapidly from mitosis to G1 phase and increased steadily throughout S phase, but the transient activation on entry into S phase was not observed. Complete inhibition of the (Na+-K+)ATPase mediated K+ influx by ouabain (5 mM) prevents the cells from entering S phase, while partial inhibition by lower concentrations of ouabain (0.2 and 0.5 mM; km = 0.17 mM) causes partial blockage in G1 and, to a lesser extent, a reduced rate of progression through the rest of the cell cycle. We conclude that the transient increase in (Na+-K+)ATPase mediated K+ influx at the G1/S transition is a prerequisite for entry into S phase, while maintenance of adequate levels of K+ influx is necessary for normal rate of progression through the rest of the cell cycle. 相似文献
12.
The mitogenic activity of somatomedin-C/insulinlike growth factor-I (SM-C/IGF-I) appears to be greatly influenced by cell culture conditions, especially the presence of other growth factors and nutrients in the culture medium. To investigate the effect of cell density on SM-C/IGF-I activity, we have evaluated SM-C/IGF-I binding and stimulation of DNA synthesis and cell replication as a function of cell density in cultured human fibroblast monolayers. At fibroblast concentrations of 2.7 X 10(5) and 1.48 X 10(6) cells per 60-mm dish, specific binding of [125I]SM-C/IGF-I per 10(6) cells was 170% higher in sparse than dense monolayers (9.3% vs. 3.4%). Increased binding in sparse monolayers was attributable to approximately twice as many receptors in sparse as in dense cells (31,000 vs. 16,000 sites per cell), as well as to a modest increase in the affinity constant. Similarly, half-maximal stimulation of [methyl-3H]thymidine incorporation was achieved at SM-C/IGF-I concentrations of 2.5 ng/ml in sparse cells but required 20 ng/ml in dense cells. Although this required only 45% occupancy of membrane receptors on sparse cells, and almost 80% occupancy on dense cells, the total number of occupied receptors was similar in both sparse and dense cells (approximately 13,000 receptors/cell for half-maximal stimulation). The presence of increased numbers of "functional receptors" on sparse fibroblasts thus results in enhanced sensitivity to SM-C/IFG-I stimulation of DNA synthesis and cell replication. Progressive decreases in the number of functional receptors, secondary to cell crowding, may contribute to density-dependent inhibition of fibroblast growth. 相似文献
13.
Control of lipoprotein lipase secretion in mouse macrophages 总被引:2,自引:0,他引:2
The regulation of secretion of lipoprotein lipase (LPL) was studied in in vitro-derived mouse bone marrow macrophages (BMM), peritoneal exudate and resident macrophages and in the macrophage-like tumor cell line J774.1. BMM in cultures initiated with low concentrations of bone marrow cells (LC-BMC cultures) secrete more LPL per cell than BMM in cultures initiated with high concentrations of bone marrow cells (HC-BMC cultures). The suppressed state of LPL secretion in HC-BMC cultures could be alleviated by the addition of a colony-stimulating factor source (L-cell-conditioned medium; L-CM) onto the culture medium or exchanging the medium of HC-BMC cultures with medium from LC-BMC cultures for short periods (4 h). Addition of L-CM increased LPL secretion also in LC-BMC cultures. Addition of L-CM to fresh culture medium had little or no effect, suggesting that, in addition to requirement for L-CM, optimal expression depended also on factors released by the growing cells, probably providing optimal growth conditions. L-CM enhanced LPL secretion by thioglycollate-elicited peritoneal macrophages and had no effect on LPL secretion by resident peritoneal macrophages. Secretion of LPL from adherent J774.1 cells showed a biphasic effect. Secretion increased with cell density up to the point when growth inhibition was observed. In dense cultures in which cell proliferation was almost arrested, LPL secretion was remarkably suppressed (80-90%). Change of medium of dense cultures to fresh medium or medium conditioned by sparse cultures (for the last 4 h of culture) led to enhancement of LPL secretion to levels similar to those optimally expressed by sparse cultures. L-CM did not enhance LPL secretion from J774.1 cells. Dense cultures of both BMM and J774.1 cells did not contain a stable inhibitor of LPL secretion and medium from sparse cultures did not contain an inducer of LPL secretion. The data suggest that proliferating macrophages secrete large amounts of LPL, whereas in nonproliferating, quiescent cells, this activity is much reduced. L-CM enhances LPL secretion in quiescent BMM and peritoneal exudate cells to levels expressed by proliferating cells. Since this effect is already expressed after a 4 h incubation period, it is not dependent on cell cycling but could be one of the early responses to this macrophage mitogen. In J774.1 cells, a change of medium is a sufficient signal for enhancement of LPL secretion in quiescent cells. 相似文献
14.
Ouabain is a specific inhibitor of sodium, potassium-dependent adenosine triphosphatase (Na,K-ATPase), a P-type ion-transporting ATPase which is essential for the maintenance of adequate concentrations of intracellular Na+ and K+ ions. The present study describes the establishment of a ouabain-resistant mutant, TLouaR, from a human trophoblast cell line TL. Morphologically TL and TLouaR are indistinguishable, but, TLouaR is about 1000 times more resistant to the cytotoxic effect of ouabain and > 2000 times to that of bufalin and yet ouabain can retard the growth of the TLouaR cells and in parallel reduce its cloning efficiency in a time- and dose-dependent manner. Furthermore, Na,K-ATPase activity from TLouaR cells is inhibitable by ouabain albeit with lower efficiency. [3H]ouabain binding studies reveal that TLouaR cells have less (P < 0.05) ouabain binding sites (1.7 +/- 0.15 x 10(4)/cell vs. 2.3 +/- 0.115 x 10(4)/cell in the control). However, affinities (dissociation constants Kd) to ouabain for TL and TLouaR cells are not significantly different. Lastly, Na,K-ATPase activity (1.375 +/- 0.25 micromole ATP/min mg protein) of TLouaR cells is significantly higher (P < 0.05) than that of the TL cells (0.895 +/- 0.12 micromole ATP/min x mg protein). These studies show that the interactions between ouabain and Na,K-ATPase can be mediated through different pathways resulting in diverse phenotypic characteristics. In addition, ouabain resistance does not necessarily reflect the lack of response to the digitalis drug. The exact mechanisms of ouabain resistance observed in the present study remain to be determined but the TLouaR cells may be the best tool to uncover the many functional characteristics of Na,K-ATPase. 相似文献
15.
Brodsky V Zvezdina N Nechaeva N Novikova T Gvasava I Fateeva V Gracheva H 《Cell biology international》2003,27(11):935-942
Pretreatment of hepatocyte cultures with 1 microM d-l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol-HCL (PPPP) for 24 h decreased the ganglioside GM1 content of the cells by approximately 50% and that of the conditioned medium by 90%. No rhythm in the rate of protein synthesis was detected in dense cultures pretreated with PPPP, but was observed in control dense cultures. Conditioned medium from control dense cultures induced synchrony in sparse cultures, which were non-synchronous in their own medium. In contrast, conditioned medium from dense cultures pretreated with PPPP did not synchronize sparse cultures. Since protein synthesis rhythm is a marker of cell synchronization, i.e. their co-operative activity, then non-oscillatory behavior means loss of cell co-operation. The protein synthesis rhythm was restored 24 h after hepatocytes were transferred to PPPP-free medium. Restoration was more rapid when 0.9 microM gangliosides (standard mixture from bovine brain) were added to the medium just after the withdrawal of PPPP. These novel results concerning the loss of rhythm of protein synthesis in low GM1 ganglioside medium support the conclusion that ganglioside is implicated in the regulation of cell co-operative activity. 相似文献
16.
Energetic mechanism of system A amino acid transport in normal and transformed mouse fibroblasts 总被引:2,自引:0,他引:2
Ouabain treatment (0.4 mM) of normal and transformed C3H-10T1/2 cells caused a progressive increase in 2-aminoisobutyrate (AIB) transport reaching a maximum after 16 to 18 h exposure. There was a virtually complete blockage of this stimulated rate when 3 microM cycloheximide (CHX) was added together with ouabain at T = 0. In the transformed cell, addition of CHX after 14 h had no effect; in the normal cell, it inhibited (ca. 50%) the final AIB transport rate achieved after 24 h. The t1/2 for reaching maximal activity (insensitive to CHX exposure) was thus shifted from 8 h in the transformed cell to 15 h in the normal cell. Since the rate of achieving maximal activity in the absence of CHX was about the same in the two cells, the shift in t1/2 in the presence of CHX suggests that the rate of degradation is more rapid in the normal cell. Following ouabain treatment, the apparent Km for Na+ was decreased in both cells. The Km returned to the basal level 1 h after ouabain removal in the normal cell, but remained low in the transformed cell during this time period. The stimulation of AIB transport following ouabain removal was largely abolished by a proton ionophore (1799), a lipophilic cation (tetraphenyl-phosphonium), or ouabain. These results suggest that, under the conditions of ouabain stress, there is a switch in the bioenergetic mechanism. The Na+/K+ pump and System A transporter appear to be linked and the membrane potential generated by the Na+/K+ pump activity becomes a major driving force for AIB uptake. 相似文献
17.
(Na,K)-ATPase and Ouabain binding in reticulocytes from dogs with high K and low K erythrocytes and their changes during maturation 总被引:2,自引:0,他引:2
The present study demonstrated that dog reticulocytes had considerable amounts of (Na,K)-ATPase, but lost it rapidly during maturation into erythrocytes. Furthermore, reticulocytes from dogs possessing erythrocytes characterized with high (Na,K)-ATPase activity and high K, low Na concentrations (HK dogs; Maede, Y., Inaba, M., and Taniguchi, N. (1983) Blood 61,493-499) had more ouabain binding sites than cells from normal dogs (LK dogs). Our results were as follows: i) The maximal binding capacities (Bmax) for ouabain binding at equilibrium were approximately 0 and 1,500 binding sites/cell in LK and HK dog erythrocytes, respectively. ii) Reticulocytes from LK dogs possess approximately 5,700 ouabain binding sites/cell. iii) The Bmax value for ouabain in HK reticulocytes was about 10,000 sites/cell, being 2-fold that in LK reticulocytes. iv) Ouabain-sensitive fluxes of 24Na and 42K in each type of reticulocyte were compatible with the number of ouabain binding sites on the cells. v) Ouabain binding capacity, as well as (Na,K)-ATPase activity, in the reticulocytes from LK dogs fell rapidly to nearly zero during the maturation into erythrocytes. vi) Although reticulocytes from HK dogs also showed a similar regression of (Na,K)-ATPase during maturation, they retained a certain number of ouabain binding sites even after maturation, resulting in the high activity of (Na,K)-ATPase in HK erythrocyte membrane. 相似文献
18.
Na(+),K(+)-ATPase, a basolateral transporter responsible for tubular reabsorption of Na(+) and for providing the driving force for vectorial transport of various solutes and ions, can also act as a signal transducer in response to the interaction with steroid hormones. At nanomolar concentrations ouabain binding to Na(+),K(+)-ATPase activates a signaling cascade that ultimately regulates several membrane transporters including Na(+),K(+)-ATPase. The present study evaluated the long-term effect of ouabain on Na(+),K(+)-ATPase activity (Na(+) transepithelial flux) and expression in opossum kidney (OK) cells with low (40) and high (80) number of passages in culture, which are known to overexpress Na(+),K(+)-ATPase (Silva et al., 2006, J Membr Biol 212, 163-175). Activation of a signal cascade was evaluated by quantification of ERK1/2 phosphorylation by Western blot. Na(+),K(+)-ATPase activity was determined by electrophysiological techniques and expression by Western blot. Incubation of cells with ouabain induced activation of ERK1/2. Long-term incubation with ouabain induced an increase in Na(+) transepithelial flux and Na(+),K(+)-ATPase expression only in OK cells with 80 passages in culture. This increase was prevented by incubation with inhibitors of MEK1/2 and PI-3K. In conclusion, ouabain-activated signaling cascade mediated by both MEK1/2 and PI-3K is responsible for long-term regulation of Na(+) transepithelial flux in epithelial renal cells. OK cell line with high number of passages is suggested to constitute a particular useful model for the understanding of ouabain-mediated regulation of Na(+) transport. 相似文献
19.
Inhibition of Na+,K+-ATPase by ouabain triggers epithelial cell death independently of inversion of the [Na+]i/[K+]i ratio 总被引:1,自引:0,他引:1
Pchejetski D Taurin S Der Sarkissian S Lopina OD Pshezhetsky AV Tremblay J deBlois D Hamet P Orlov SN 《Biochemical and biophysical research communications》2003,301(3):735-744
Treatment with ouabain led to massive death of principal cells from collecting ducts (C7-MDCK), indicated by cell swelling, loss of mitochondrial function, an irregular pattern of DNA degradation, and insensitivity to pan-caspase inhibitor. Equimolar substitution of extracellular Na(+) by K(+) or choline(+) sharply attenuated the effect of ouabain on intracellular Na(+) and K(+) content but did not protect the cells from death in the presence of ouabain. In contrast to ouabain, inhibition of the Na(+)/K(+) pump in K(+)-free medium increased Na(+)(i) content but did not affect cell survival. In control and K(+)-free medium, ouabain triggered half-maximal cell death at concentrations of approximately 0.5 and 0.05 microM, respectively, which was consistent with elevation of Na(+)/K(+) pump sensitivity to ouabain in K(+)-depleted medium. Our results show for the first time that the death of ouabain-treated renal epithelial cells is independent of the inhibition of Na(+)/K(+) pump-mediated ion fluxes and the [Na(+)](i)]/[K(+)](i) ratio. 相似文献
20.
F.Arthur McMorris 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1983,762(4):560-568
Specific activity of the myelin enzyme, 2′:3′-cyclic-nucleotide 3′-phosphohydrolase (EC 3.1.4.37), increases 2- to 10-fold when sparsely inoculated cultures of C6 rat glioma cells are allowed to grow to high cell density. Cyclic-nucleotide phosphohydrolase specific activity is also induced in C6 cells and in oligodendrocytes by dibutyryl cyclic AMP or by agents that elevate intracellular cyclic AMP. In this report, we have compared the density-dependent induction of cyclic-nucleotide phosphohydrolase activity with the cyclic AMP-dependent induction. Dibutyryl cyclic AMP induced cyclic-nucleotide phosphohydrolase specific activity in both sparse and dense cultures which had very different density-dependent cyclic-nucleotide phosphohydrolase activities. Induction of both cyclic-nucleotide phosphohydrolase specific activity and intracellular cyclic AMP content by norepinephrine also occurred to a similar degree in sparse and dense cultures. Similar results were obtained for several clones of C6 cells, and for a clone of oligodendrocyte x C6 cell hybrids. Induction of cyclic-nucleotide phosphohydrolase by norepinephrine or dibutyryl cyclic AMP was not due to a change in cell density or rate of cell proliferation, nor did cell density have any appreciable effect on cyclic AMP content of the cells. These results show that regulation of cyclic-nucleotide phosphohydrolase activity in C6 cells involves two distinct mechanisms. 相似文献