摘要:

AbstractThe effect of hemicholinium‐3 (HC‐3) on choline uptake and phosphatidylcholine (PC) biosynthesis was examined in human leukemic monocyte‐like U937 cells. HC‐3 inhibited [3H]choline uptake in a dose‐ and time‐dependent manner. After a 3 h treatment, HC‐3 (100 μM) decreased choline uptake by as much as 80 per cent (p<0·0001;n= 4). Reduction of incorporation of label into PC was also detected in a dose‐dependent manner; the extent of inhibition, however, was always 10–20 per cent less than that observed in the total uptake. At 3 h HC‐3 decreased the incorporation into PC by 65 per cent (p<0·0001;n= 5). Kinetic studiesin vivoshowed that HC‐3 inhibited total uptake and incorporation into PC differently, suggesting that the labelling of PC is not simply dictated by [3H]choline uptake. In separate experiments, cells were pretreated with 100 μM HC‐3 for 3 h. After washing, the inhibitory effect on total uptake was no longer observed, while a 20 per cent stimulation of the incorporation into PC was obtained in these pretreated cells. In pulse‐chase studies, the cells were prelabelled with [3H]choline for 30 min and chased with HC‐3 for up to 3 h; the results showed a significant stimulation of incorporation into PC in a longer chase with 100 μM HC‐3. After a 3 h treatment, the cytosolic CTP:cholinephosphate cytidylytransferase (CT) was activated by 56 per cent, while choline kinase (CK) was inhibited slightly. The stimulation of CT was not simply due to the intact HC‐3 molecule, and there was no redistribution of CT between cytosol and microsomes. Taken together, the results suggest that HC‐3 activates PC biosynthesis apart from t

ISSN:0263-6484    

DOI:10.1002/cbf.290120202

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThe effect of lidocaine on [3H]choline uptake and the incorporation of label into phosphatidylcholine (PC) in human monocyte‐like U937 cells was investigated. Lidocaine inhibited the rate of choline uptake in a dose‐dependent manner; at 3·2 mMit resulted in a drastic reduction, by as much as 65 per cent (n= 10;p<0·0005) or 55 per cent (n= 10;p<0·0006) in a 3‐ or 6‐h incubation, respectively. Lidocaine also decreased the rate of choline incorporation into PC in a dose‐dependent manner. At the highest dose, nearly 70 per cent or 45 per cent reduction was seen in a 3‐ or 6‐h incubation, respectively. Analysis of choline‐containing metabolites showed that the major label association with phosphocholine and PC was reduced to a similar extent which was also parallel to the inhibition of choline uptake. At 3·2 mMlidocaine, the reduction of choline uptake was shown to follow a competitive inhibition. In the case of [3H] choline incorporation into PC, the inhibitory pattern was shown to be of a mixed type. The pulse‐chase study dissecting the effect on choline metabolism from that on total choline uptake indicated that lidocaine exerted an additionally inhibitory effect on intracellular choline metabolism into PC. In a separate protocol in which the labelled cells were first allowed to be chased until3H‐incorporation into PC reached a steady state, lidocaine no longer showed any effect. These results seem to exclude the possibility of enhanced PC breakdown and further suggest that the main inhibitory effect is on the CDP‐choline pathway for PC biosynthesis. After a 3‐h treatment, CTP: cholinephosphate cytidylyltransferase (CYT) in both the cytosolic and microsomal fractions was inhibited by approximately 20 per cent, while choline kinase (CK) and choline phosphotransferase (CPT) remain relatively unchanged. There was no evidence for translocation of CYT between cytosol and microsomes. Taken together, we have demonstrated a dual inhibitory function of lidocaine which inhibits PC biosynthesis in addition to its ability to block choline upta

ISSN:0263-6484    

DOI:10.1002/cbf.290120203

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThe regulation of intracellular creatine concentration in mammalian cells is poorly understood, but is thought to depend upon active sodium‐linked uptake of creatine from extracellular fluid. In normal human erythrocytes, creatine influx into washed cells was inhibited by 40 per cent in the absence of extracellular sodium. In washed cells from uraemic patients, sodium‐independent creatine influx was normal, whereas the sodium‐dependent component of creatine influx was 3·3 times higher than normal, possibly relecting the reduced mean age of uraemic erythrocytes. In spite of this, the intracellular creatine concentration was no higher than normal in uraemic erythrocytes, implying that some factor in uraemic plasmain vivoinhibits sodium‐dependent creatine influx. Both in normal and uraemic erythrocytes, the creatine concentration was 10 times that in plasma, and the concentration in the cells showed no detectable dependence on that in plasma, suggesting that the intracellular creatine concentration is controlled by an active saturable process. Active sodium‐dependent accumulation of creatine was also demonstrated in L6 rat myoblasts and was inhibited when transport was measured in the presence of 10−4Mouabain or digoxin, implying that uptake was driven by the transmembrane sodium gradient. However, when creatine influx was measured immediately after ouabain or digoxin had been washed away, it was higher than in control cells, suggesting that Na,K‐ATPase and/or sodium‐linked creatine transport are up‐regulated when treated with inhibit

ISSN:0263-6484    

DOI:10.1002/cbf.290120204

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractTotal energy production in rabbit reticulocytes amounted to 136·52 ± 6·50μmol ATP h−1ml−1of reticulocytes: 88·3 per cent was provided by oxidative phosphorylation, whereas only 11·7 per cent by aerobic glycolysis. Na+K+‐ATPase accounted for 23 per cent, i.e. 27·65 ± 2·55μmol ATP h−1ml−1of reticulocytes, in the overall energy consumption in reticulocytes of rabbits. Under basal conditions ATP for Na+K+‐ATPase activity was derived exclusively from oxidative phosphorylation. However, when the activity of Na+K+‐ATPase was increased due to the stimulation of adenylate cyclase by (−)‐isoprenaline, the additional energy required was provided by aerobic glycolysis. These results indicate that two different compartments, one cytosolic and the other mitochondrial, provide energy for Na+K+‐ATP

ISSN:0263-6484    

DOI:10.1002/cbf.290120205

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThe effects of omeprazole, a proton pump inhibitor, on gene expression, protein synthesis, intracellular storage and secretion of pepsinogen in guinea pig stomach were investigated.After treatment with omeprazole for five days, acid and pepsinogen secretion into the gastric lumen was significantly reduced. Concomitant with this, there was an increase in intracellular pepsinogen as demonstrated by increased pepsin activity in the gastric mucosa, more intense immunohistochemical staining by antibodies specific of pepsinogen and accumulation of secretory granules in the cells producing pepsinogen. In these cells, the amount for pepsinogen mRNA was reduced as revealed by Northern blotting andin situhybridization. Ultrastructurally the endoplasmic reticulum of these cells was poorly developed, the findings being consistent with a reduction in protein synthesis. It appears that omeprazole inhibits the secretion of pepsinogen, increasing the intracellular store and leading to the reduction in gene expression probably by a feedback mechanism and consequent reduction in pepsinogen synthesis. Since these changes were most evident in the acid‐secreting fundic gland mucosa, as compared with other mucosae secreting only pepsinogen, namely pyloric and duodenal mucosa, it appears probable that these changes are linked with omeprazole‐induced reduction in the acid secret

ISSN:0263-6484    

DOI:10.1002/cbf.290120206

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThe intracellular effect of dexamethasone (DXME) on the activity and gene expression of ornithine decarboxylase (ODC) was studied in Syrian hamster embryo cells (SHE). The ODC activity (expressed as nmoles decarboxylated ornithine mg−1protein h−1) was 4·61 ± 0·14 in untreated cells, whereas it increased to 14·38 ± 0·26 after 5h treatment with 1·6 × 10−7MTPA. In contrast, DXME (2·5 × 10−5M) reduced the ODC activity by 50 per cent to 2·35 ± 0·22. In cells co‐treated for 5 h with TPA and DXME, ODC acitivity decreased to the level of the untreated cells. However, when DXME was added 3 h after TPA treatment for 2 h, in the continuous presence of TPA, the ODC activity unexpectedly increased further to 16·44 ± 1·05. The modulation of ODC activity correlated partly with the level of ODC mRNA. Thus when cells were treated with TPA, and ODC mRNA increased threefold, whereas it decreased by 30 per cent when the cells were exposed to DXME. In TPA–DXME co‐treated cells, as in TPA pretreated cells followed by DXME for 2 h, a decrease (31·25 per cent and 12·5 per cent respectively) was observed in ODC mRNA. In turnover studies, DXME was found to increase the stability of ODC; the discrepancy between ODC activity and ODC mRNA levels could result from an inhibitory effect of the corticoid on proteolysis of ODC.Studies of lysosomal protease showed that the activities of cathepsins L, B and H decreased following TPA treatment. DXME also inhibited cathepsin L and B activities, but stimulated cathepsin H. Analysis of neutral cytosolic protease activity by gelatin and casein zymograms showed that TPA strongly stimulated the activity of a 70 kDa gelatinase. DXME was able to inhibit the induction of such cytosolic proteases under all the treatment conditions. When the cytosolic protein was incubatedin vitro, at 37°C, in the presence of 2 mMCaCl2, the ODC activity decreased by 50 per cent after 30 min incubation. Further decrease was achieved whenp‐amino‐phenylmercuric acetate, a protease activator, was added. Proteolytic activity was not inhibited after the addition of 10 μg ml−1of TIMP‐1. In contrast, the addition of EDTA restored the ODC activity completely.We postulate that modification of the 70 kDa cytosolic metalloprotease activity could interact with t

ISSN:0263-6484    

DOI:10.1002/cbf.290120207

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractWe have investigated whether or not ATP or other nucleoside di‐ and trisphosphates (including some nonhydrolysable ATP analogues) can stimulate the activity and/or the processivity of DNA polymerase α associated with the nuclear matrix obtained from HeLa S3 cell nuclei that had been stabilized at 37°C prior to subfractionation, as has been reported previously for DNA polymerase α bound to the nuclear matrix prepared from 22‐h regenerating rat liver. We have found that HeLa cell matrix‐associated DNA polymerase α activity could not be stimulated at all by ATP or other nucleotides, a behaviour which was shared also by DNA polymerase α activity that solubilizes from cells during the isolation of nuclei and that is thought to be a form of the enzyme not actively engaged in DNA replication. Moreover, the processivity of matrix‐bound DNA polymerase α activity was low (<10 nucleotides). These results were obtained with the matrix prepared with either 2MNaCl or 0·25M(NH4)2SO4and led us to consider that a 37° incubation of isolated nuclei renders resistant to high‐salt extraction a form of DNA polymerase α which is unlikely to be involved in DNA

ISSN:0263-6484    

DOI:10.1002/cbf.290120208

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractThe effects of either radiation or hyperthermia on the differentiation potential of NG108‐15, a neuroblastoma‐glioma hybrid cell line, were studied. After radiation and hyperthermia, the outgrowth of neurites from NG108‐15 cells was potentiated, and polarizing current and voltage pulses induced a distinct action potential and a diphasic (inward following outward) current, respectively. An increase in the specific activity of acetylcholinesterase was also observed. In addition, both treatments induced an elevation of the concentration of intracellular calcium in some cells. The increase in intracellular calcium concentration caused by applying the calcium ionophore, A23187, induced differentiation. It is suggested that both the radiation‐ and the hyperthermia‐induced increases of electrical excitability and acetylcholinesterase activity may have originated from an increase in intracellular Ca2+conc

ISSN:0263-6484    

DOI:10.1002/cbf.290120209

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

Abstract4‐Hydroxynonenal (HNE) degradation was investigated in isolated perfused rat hearts of the WKY and SHR strains before and after ischemia. HNE (10 μmoles l−1) were infused and the concentration of HNE in the effluent was determined. The rate of initial consumption was about 50 nmoles min−1g−1wet weight in hearts of both the WKY and SHR rats. In the WKY rat hearts, this rate of HNE degradation did not change during several minutes of HNE infusion and also remained constant during postischemic reperfusion. In the hearts of the SHR rats the HNE degradation rate declined within 5 min to 25 nmoles min−1g−1wet weight. Also during postischemic reperfusion, there was a lower HNE degradation rate in the SHR rat hearts than in the WKY rat hearts. The influence of hypertrophy on the rate of HNE degradation is discussed. It is suggested that the low degradation of the cytotoxic lipid peroxidation product, HNE, in hypertrophic hearts may contribute to reduced antioxidant defence in

ISSN:0263-6484    

DOI:10.1002/cbf.290120210

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY

摘要:

AbstractFerricyanide reductase activity of plasma membranes isolated from Ehrlich ascites tumour cells was very sensitive to trypsin treatment. The decreases of activity observed after treatment with different glycosidases suggests that ferricyanide reductase is a glycoprotein. The opposite effects of phospholipase A2and phospholipase C on the redox activity indicate that the phospholipidic environment plays an important role in the function of ferricyanide reductase. Sodium ions at millimolar concentrations, and some divalent cations at micromolar concentrations (Ca2+, Mg2+, Sr2+, and Mn2+) behaved as stimulators of ferricyanide reductase activity.

ISSN:0263-6484    

DOI:10.1002/cbf.290120211

出版商:John Wiley&Sons, Ltd.    

年代:1994

数据来源: WILEY