摘要:

AbstractOxidative stress induced by hydrogen peroxide (H2O2) may contribute to the pathogenesis of ischemic‐reperfusion injury in the heart. For the purpose of investigating directly the injury potential of H2O2on heart muscle, a cellular model of H2O2‐induced myocardial oxidative stress was developed. This model employed primary monolayer cultures of intact, beating neonatal‐rat cardiomy‐ocytes and discrete concentrations of reagent H2O2in defined, supplement‐free culture medium. Cardiomyocytes challenged with H2O2readily metabolized it such that the culture content of H2O2diminished over time, but was not depleted. The consequent H2O2‐induced oxidative stress caused lethal sarcolemmal disruption (as measured by lactate dehydrogenase release), and cardiomyocyte integrity could be preserved by catalase. During oxidative stress, a spectrum of cellular derangements developed, including membrane phospholipid peroxidation, thiol oxidation, consumption of the major chain‐breaking membrane antiperoxidant (α‐tocopherol), and ATP loss. No net change in the protein or phospholipid contents of cardiomyocyte membranes accompanied H2O2‐induced oxidative stress, but an increased turnover of these membrane constituents occurred in response to H2O2. Development of lethal cardiomyocyte injury during H2O2‐induced oxidative stress did not require the presence of H2O2itself; a brief “pulse” exposure of the cardiomyocytes to H2O2was sufficient to incite the pathogenic mechanism leading to cell disruption. Cardiomyocyte disruption was dependent upon an intracellular source of redox‐active iron and the iron‐dependent transformation of internalized H2O2into products (e.g., the hydroxyl radical) capable of initiating lipid peroxidation, since iron chelators and hydroxyl‐radical scavengers were cytoprotective. The accelerated turnover of cardiomyocyte‐membrane protein and phospholipid was inhibited by antiperoxidants, suggesting that the turnover reflected molecular repair of oxidized membrane constituents. Likewise, the consumption of α‐tocopherol and the oxidation of cellular thiols appeared to be epiphenomena of peroxidation. Antiperoxidant interventions coordinately abolished both H2O2‐induced lipid peroxidation and sarcolemmal disruption, demonstrating that an intimate pathogenic relationship exists between sarcolemmal peroxidation and lethal compromise of cardiomyocyte integrity in response to H2O2‐induced oxidative stress. Although sarcolemmal peroxidation was causally related to cardiomyocyte disruption during H2O2‐induced oxidative stress, a nonperoxidative route of H2O2cytotoxicity was also identified, which was expressed in the complete absence of cardiomyocyte‐membrane peroxidation. The latter mode of H2O2‐induced cardiomyocyte injury involved ATP loss such that membrane peroxidation and cardiomyocyte disruption on the one hand and cellular de‐energization on the other could be completely dissociated. The cellular pathophysiology of H2O2as a vectorial signal for cardiomyocyte necrosis that “triggers” irreversible peroxidative disruption of the sarcolemma has implications regarding potential mecha

ISSN:0021-9541    

DOI:10.1002/jcp.1041490302

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractIs an intact plasma membrane responsible for keeping hemoglobin and water within the human erythrocyte? If not, what is responsible? How free is Hb to move about within the erythrocyte? To answer these questions erythrocytes were taken for phase contrast microscopy, transmission electron microscopy (TEM), determination of water‐holding capacity, and proton NMR studies both before and after membrane disruption with a nonionic detergent (Brij 58). Addition of 0.2% Brij to a D2O saline solution of hemoglobin (Hb) caused particles of Hb to appear and to aggregate. This aggregation of Hb caused the amplitude of the Hb proton NMR spectra to decrease. Thus, the less mobile the Hb the lower the Hb proton spectra amplitude. Erythrocytes washed in D2O saline showed proton NMR spectra of relatively low amplitude. Addition of Brij (0.2%) to these erythrocytes caused increased Hb mobility within these erythrocytes. The TEM of fixed and thin‐sectioned erythrocytes treated with Brij showed disruption of the plasma membrane of all erythrocytes regardless of whether or not they had lost Hb. Brij‐permeabilized erythrocytes washed in D2O saline or in a D2O K buffer maintained a higher heavy water‐holding capacity upon centrifugation as compared to nonpermeabilized erythrocytes. The TEM of Brij‐treated and washed erythrocyte “shells” revealed a continuous submembrane lamina but no other evidence of cytoskeletal elements. The water‐holding capacity of the erythrocyte can be accounted for by the water‐holding capacity of hemoglobin. The evidence favors a relatively immobile state of Hb and of water in the erythrocyte that is not immediately dependent on an intact plasma membrane but is attributed to interactions between Hb molecules and the s

ISSN:0021-9541    

DOI:10.1002/jcp.1041490303

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractExtending our previous observation that tissue transglutaminase (TGase) binds to extracellular matrix (ECM) fibronectin, we report here that endogenous tissue TGase is localized on the adjacent ECM after puncture wounding embryonic human lung fibroblasts (WI‐38). The bound TGase persisted at the wound site for many hours, demonstrated by immunofluorescence and by catalytic activity using an overlay assay. The binding characteristics of TGase with ECM were studied further by the addition of exogenous TGase to cell monolayers and monitoring by immunofluorescence or overlay catalytic activity assays. Binding occurred equally well at 4°C or 37°C. Prior incubation of exogenous TGase with guanosine 5′‐triphosphate (GTP), guanosine 5′‐diphosphate (GDP), or adenosine triphosphate (ATP) had little effect on the amount bound to matrix, but prior treatment with calcium, magnesium, strontium, or manganese ions enhanced binding 2‐to 3‐fold. The Ca++‐dependent change was a concentration‐dependent effect on soluble exogenous TGase, rather than an effect on ECM. Immunofluorescent techniques showed that binding of exogenous TGase to ECM was prevented by prior mixing with fibronectin or collagen, but not with several other ECM components, including laminin, elastin, chondroitin sulfate, heparan sulfate, and hyaluronic acid. ECM‐bound TGase was released by 2 M potassium thiocyanate (KSCN) treatment but was not released by treatment with a variety of amino acids, salts, reducing agents, glycerol, or ot

ISSN:0021-9541    

DOI:10.1002/jcp.1041490304

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe components of45calcium (Ca) uptake were studied in saponin skinned rat caudal artery. The steady‐state Ca content increased when the free Ca concentration was varied from 10−8to 10−4M but was reduced by azide when the free Ca concentration exceeded 3.1 μM. The azide sensitivity and low affinity for Ca were consistent with functional mitochondria. The azide‐insensitive component consisted of a small bound and a larger releasable Ca fraction. After skinning in Triton X‐100, approximately 4 μmol Ca/kg wet tissue remained, which represented a tightly bound but slowly exchangeable Ca pool. The Ca content was independent of the free Ca concentration and MgATP, and it was not released with A‐23187 or Ca. The Ca content of the larger fraction was a higher order function of the free Ca concentration and was released with A‐23187, indicating it resided within a membrane‐bounded structure. Ca uptake by the releasable fraction was increased by oxalate, MgATP, phosphocreatine, temperature, phosphate, and ruthenium red and represents Ca sequestered by the sarcoplasmic reticulum (SR) with little contribution from other Ca binding or storage sites. It is described by the coefficients Umax= 96.94 μmol/kg wet tissue, K1/2= 0.75 μM, and Hill coefficient = 1.70. The SR in this preparation regulates cytosolic Ca concentrations under physiological conditions and can accumulate Ca by MgATP‐dependent and MgATP‐independent processes. The larger, MgATP‐dependent Cauptake is described by the coefficients Umax= 72.87 μmol/kg wet tissue, K1/2= 0.8 μM, and Hill coefficient = 2.09 and is consistent with Ca sequestered by the Ca‐transport ATPase of smooth muscle SR. The smaller, MgATP‐independent uptake is described by the coefficients Umax= 24.14 μmol/kg wet tissue, K1/2= 0.56 μM, and Hill coefficient = 1.01 and represents Ca sequestered by an unidentified mech

ISSN:0021-9541    

DOI:10.1002/jcp.1041490305

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe mechanism of histidinol (HST) ‐induced heat protection was investigated to test the hypothesis that the cessation of protein synthesis itself is one of the events involved in heat protection. For this study, we isolated three HST‐resistant mutant strains. HST (5 mM), which inhibited protein synthesis by 88% in the wild type, caused only 0, 9, and 25% inhibition in three mutants, respectively. The drug, which afforded heat protection, (i.e., a 125‐fold increase in survival from 4 x 10−3to5 x 10−1after 2 hr at 43°C in wild type), did not protect mutant cells from heat killing. In contrast, cycloheximide (10 μg/ml) which inhibited protein synthesis by 95% in both wild type and mutant cell types, protected both cell types from heat killing. Therefore, these results suggest that the cessation of protein synthesis, per se, preventing synthesis of nascent polypeptides, is a major event leading to heat

ISSN:0021-9541    

DOI:10.1002/jcp.1041490306

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractIn RBL‐2H3 rat leukemic mast cells, cross‐linking anti‐DNP IgE‐receptor complexes with multivalent antigen (DNP‐BSA) activates a signal transduction pathway leading to Ca2+influx and secretion. Cross‐linking IgE‐receptor complexes also stimulates a pathway that inactivates (desensitizes) receptors;this pathway becomes important at high concentrations of cross‐linking antigen. Recent evidence that antigen‐induced secretion is impaired by mycophenolic acid (MPA), an inhibitor of guanine nucleotide synthesis de novo, has implicated a GTP‐binding protein (G protein) in the signaling pathway. Other recent studies have indicated that the conversion of cross‐linked receptors to a detergent‐insoluble (cytoskeleton‐associated) form at high antigen concentrations is correlated with the loss of signaling activity. Here we show that secretion elicited by an optimal concentration of antigen (0.05 μg/ml DNP‐BSA) is only inhibited by about 25% in guanine nucleotide‐depleted cells, whereas secretion elicited by 5 μg/ml DNP‐BSA, a concentration in the range that causes the high‐dose inhibition of secretion, is inhibited by more than 60%. We also show that IgE‐receptor complexes are insolubilized in response to 5 but not 0.05 μg/ml DNP‐BSA in both control and guanine nucleotide‐depleted cells. Importantly, the extent of insol‐ubilization elicited by 5 μg/ml DNP‐BSA is increased by more than 60% in the guanine nucleotide‐depleted samples. These results raise the possibility that guanine nucleotide depletion reduces the secretory response to high antigen concentrations in two ways: by inhibiting the G protein‐coupled signaling pathway and by increasing the availability of receptors to the pathway le

ISSN:0021-9541    

DOI:10.1002/jcp.1041490307

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe respective roles of H2O2and ·OH radicals was assessed from the protective effects of catalase and the iron chelator o‐phenanthroline on (1) the inhibition of protein synthesis, and (2) DNA damage and the related events (activation of the DNA repairing enzyme poly(ADP) ribose polymerase with the associated depletion of NAD and ATP stores) in cultured endothelial cells exposed to the enzyme reaction hypoxanthine‐xanthine oxidase (HX‐XO) or pure H2O2. Catalase added in the extracellular phase completely prevented all of these oxidant‐induced changes. O‐phenanthroline afforded a complete protective effect against DNA strand breakage and the associated activation of the enzyme poly(ADP) ribose polymerase. By contrast, iron chelation was only partially effective in maintaining the cellular NAD and ATP contents, as well as the protein synthetic activity. In addition, the ATP depletion following oxidant injury was much more profound than NAD depletion. These results indicate that: (1) ·OH radical was most likely the ultimate O2species responsible for DNA damage and activation of poly‐(ADP) ribose polymerase; (2) both H2O2and ·OH radicals were involved in the other cytotoxic effects (inhibition of protein synthesis and reduction of NAD and ATP stores); and (3) NAD and ATP depletion did not result solely from activation of poly(ADP) ribose polymerase, but other mechanisms are likely to be involved. These observations are also compatible with the existence of a compartmentalized intracell

ISSN:0021-9541    

DOI:10.1002/jcp.1041490308

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractA rat pheochromocytoma cell line (PC12 cells) was used as a model to investigate the role of calmodulin and its multiple mRNAs in NGF‐induced neuronal differentiation. The effect of NGF on the degree of differentiation was assayed using a simple differentiation scoring system. Significant increases in the differentiation score were seen by one day, and the scores increased about 10‐fold by 8 days of treatment. NGF also increased calmodulin in the PC12 cells; significant increases were seen by 2 days of treatment, and a maximum increase of 3‐fold was seen by 4 days. Northern blot analysis using a calmodulin riboprobe revealed that all five calmodulin mRNAs found in rat tissue were present in PC12 cells. The relative abundance of the calmodulin mRNAs was 1.7 ≥ 1.4 ≥ 2.3 ≥ 4.1 ≥ 0.9 kb. NGF treatment caused a differential increase in these mRNAs. The 1.4 kb transcript (from Gene II) was increased earlier (at 1 day) and to a greater extent (3‐fold) than any of the other mRNAs. Studies of the half‐lives (t1/2) of these mRNAs suggested that the t1/2 varied with the mRNA; the smaller the mRNA, the shorter the t1/2. However, there were no significant effects of NGF on the t1/2 of any of the mRNAs. These studies indicate that NGF elevates calmodulin in PC12 cells by causing a differential increase in the multiple mRNAs for calmodulin and that the increase in calmodulin may play some part in NGF‐induced neuronal differenti

ISSN:0021-9541    

DOI:10.1002/jcp.1041490309

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractOsteoclast activity is thought to be regulated by calcitonin, as well as by the level of ionised calcium generated locally as a result of bone resorption. The exposure of isolated osteoclasts to elevated ambient calcium levels has been shown to lower resorptive activity and to reduce rates of enzyme release. We have attempted to determine whether these effects are mediated by a divalent cation‐sensitive “calcium receptor,” as has been reported for the parathyroid chief cells. Thus, we compared the effect of alkaline earth metal cations on osteoclast function using a morphometric measure of bone resorption and a spectrophotometric method for measuring the activity of the released enzyme, acid phosphatase. The exposure of resorbing osteoclasts to between 5 and 20 mM extracellular ionised calcium ([Ca2+]e) inhibited bone resorption and enzyme release to an extent similar to that seen with 0.1 to 10 μM ionomycin. The effect of combining submaximal concentrations of [Ca2+]e(15 mM) and ionomycin (0.1 μM) resulted in additivity, suggesting that the influence of [Ca2+]eon bone resorption was mediated by elevated intracellular calcium levels ([Ca2+]e). The other cations studied (Mg2+, Ba2+) were effective and elicited similar effects, although some required higher concentrations. Thus, whilst Ca2+and Mg2+were effective at 10 to 15 mM levels, Ba2+was effective only at high (20 mM) concentrations. These findings are consistent with an influence of [Ca2+]eon osteoclast activity through an action on a surface membrane “calcium receptor” that can also bind other divalent cations, rather than by passive changes of [Ca2+]iwith [Ca2+

ISSN:0021-9541    

DOI:10.1002/jcp.1041490310

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY

摘要:

AbstractThe receptors for insulin and insulin‐like growth factor I (IGF‐I) have in common a high sequence homology and diverse overlapping functions, (e.g., the stimulation of acute metabolic events and the induction of cell growth.). In the present study, we have compared the potential of insulin and IGF‐I receptors in stimulating glucose transport activity, glucose transporter gene expression, DNA‐synthesis, and expression of proto‐oncogenec‐fosin 3T3‐L1 adipoytes which express high levels of both receptors. Binding of both hormones to their own receptors was highly specific as compared with binding to the respective other receptor (insulin receptor: KD= 3.6 nM, K1of IGF‐I>500 nM; IGF‐I receptor, KD= 1.1 nM, K1of insulin = 191 nM). Induction of proto‐oncogene c‐fos mRNA by insulin and IGF‐I paralleled their respective receptor occupancy and was thus induced by both hormones via their own receptor (EC50of insulin, 3.7; IGF‐I, 3.9 nM). Similarly, both insulin and IGF‐I increased DNA synthesis (EC50of insulin, 5.8 nM; IGF‐I, 4.0 nM), and glucose transport activity (EC50of insulin, 1.7 nM; IGF‐I, 1.4 nM), and glucose transporter (GLUT4) mRNA levels in concentrations corresponding with their respective receptor occupancy. These data indicate that in 3T3‐L1 cells the α‐subunits of insulin and IGF‐I receptors have an equal potential to stimulat

ISSN:0021-9541    

DOI:10.1002/jcp.1041490311

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1991

数据来源: WILEY