共查询到20条相似文献,搜索用时 15 毫秒
1.
R A Altschuld L M Gamelin R E Kelley M R Lambert L E Apel G P Brierley 《The Journal of biological chemistry》1987,262(28):13527-13533
The degradation and short-term resynthesis of adenine nucleotides have been examined in a preparation of isolated rat heart myocytes. These myocyte preparations are essentially free of vascular and endothelial cells, contain levels of adenine nucleotides quite comparable to those of intact heart tissue, and retain these components remarkably well for up to 2 h of aerobic incubation in the presence of 1 mM Ca2+. When the cells are rapidly and synchronously de-energized by addition of uncoupler, an inhibitor of respiration and iodoacetate, cellular ATP is degraded almost quantitatively to AMP. The AMP is then converted to either intracellular adenosine, which accumulates to high concentrations before release to the cell exterior, or to IMP. The relative contribution of these two pathways depends on the metabolic state of the cells just prior to de-energization, with IMP production favored when respiring cells are de-energized and adenosine formation predominant when glycolyzing myocytes are subjected to this treatment. Cells de-energized by anaerobiosis in the absence of glucose lose ATP and adenine nucleotides with the production of IMP and adenosine. Upon reoxygenation, these cells restore a high adenylate energy charge and about 60% of control levels of GTP. There is a net resynthesis of 5-7 nmol of adenine nucleotides.mg-1 protein with a corresponding decline in IMP. Added [14C]adenosine labels the adenine nucleotide pool, but little net resynthesis of adenine nucleotides via adenosine kinase can be detected. It therefore appears that a rapid regeneration of adenine nucleotides can occur via the enzymes of the purine nucleotide cycle in heart myocytes and is limited by the size of the IMP pool retained. 相似文献
2.
Cultured adult cardiac myocytes were exposed to anoxia under substrate-free conditions. When compared to the metabolic changes in the oxygen deficient organ, those in the anoxic cell culture proceed in a similar, yet prolonged manner. Release of cytosolic enzymes starts with minor energetic disturbances and proceeds in close correlation to the actual ATP decay. Below 2 μmol. ATP/gWW, an increasing number of cells becomes irreversibly damaged, but above, 30 min reoxygenation leads to extensive recovery of the whole preparation. The results indicate that leakage of cytosolic enzymes during the early stage of anoxia is due to a gradual protein release from the individual cells, related to reversible membrane alterations. 相似文献
3.
Shiraishi J Tatsumi T Keira N Akashi K Mano A Yamanaka S Matoba S Asayama J Yaoi T Fushiki S Fliss H Nakagawa M 《American journal of physiology. Heart and circulatory physiology》2001,281(4):H1637-H1647
Recent studies have suggested that apoptosis and necrosis share common features in their signaling pathway and that apoptosis requires intracellular ATP for its mitochondrial/apoptotic protease-activating factor-1 suicide cascade. The present study was, therefore, designed to examine the role of intracellular energy levels in determining the form of cell death in cardiac myocytes. Neonatal rat cardiac myocytes were first incubated for 1 h in glucose-free medium containing oligomycin to achieve metabolic inhibition. The cells were then incubated for another 4 h in similar medium containing staurosporine and graded concentrations of glucose to manipulate intracellular ATP levels. Under ATP-depleting conditions, the cell death caused by staurosporine was primarily necrotic, as determined by creatine kinase release and nuclear staining with ethidium homodimer-1. However, under ATP-replenishing conditions, staurosporine increased the percentage of apoptotic cells, as determined by nuclear morphology and DNA fragmentation. Caspase-3 activation by staurosporine was also ATP dependent. However, loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bax translocation, and cytochrome c release were observed in both apoptotic and necrotic cells. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition, attenuated staurosporine-induced apoptosis and necrosis through the inhibition of DeltaPsi(m) reduction, cytochrome c release, and caspase-3 activation. Our data therefore suggest that staurosporine induces cell demise through a mitochondrial death signaling pathway and that the presence of intracellular ATP favors a shift from necrosis to apoptosis through caspase activation. 相似文献
4.
C C Glembotski C E Irons A B Sprenkle C A Sei 《Canadian journal of physiology and pharmacology》1991,69(10):1525-1536
In contrast to most other endocrine peptides ANF is stored in the heart as part of a larger prohormone, often called pro-ANF, yet is found in the circulation as a 28 amino acid peptide, called ANF. It has been shown that the conversion of the 126 amino acid pro-ANF to ANF occurs in the heart. This paper summarizes studies from our laboratory that have used a primary neonatal rat heart cell culture system to investigate the location and mechanism of this relatively unusual processing event. We have found that in culture the maintenance of the cells in a glucocorticoid-containing serum-free medium is required to observe processing as occurs in vivo. The cells contain the prohormone while ANF accumulates in the medium. Various experiments with protease inhibitors, pulse-chase biosynthetic labeling, incubation of cells with ANF-related peptides, and enrichment of cultures for myocytes have resulted in our conclusion that the processing of pro-ANF takes place most likely within the cardiac myocyte just prior to, but in concert with secretion. We have expanded on the use of this processing-competent atrial myocyte culture system to investigate mechanisms of stimulated ANF secretion. It has been shown that the activation of several phospholipase C-coupled receptors (e.g., alpha 1-adrenergic and endothelin receptors) produces a robust release of ANF, but only in cultures that have been maintained under appropriate conditions. Further, it is apparent that the phenylephrine- or endothelin-mediated release of ANF depends in part on influx of extracellular calcium (Ca2+o), while the remaining component of stimulated release may depend on mobilization of intracellular calcium. It also appears that these agonists produce an initial phase of stimulated release, occurring within the first 5 min of agonist exposure, independent of Ca2+o, and a sustained phase that persists as long as the agonists remain on the cells, and depends on the presence of Ca2+o and thus calcium influx. Taken together our studies indicate that the hormonal environment may be an important factor directing the development of differentiated endocrine functions by atrial myocytes and may be involved in the regulation of ANF expression, biosynthesis, and secretion. 相似文献
5.
ATP stimulates inositol phosphates accumulation and calcium mobilization in a primary culture of rat aortic myocytes 总被引:8,自引:0,他引:8
S Phaneuf P Berta J Casanova J C Cavadore 《Biochemical and biophysical research communications》1987,143(2):454-460
The effects of extracellular ATP on phosphoinositide metabolism and intracellular Ca2+ concentration were studied in a primary culture of rat aortic myocytes. ATP increases the level of inositol phosphates, the putative second messenger for Ca2+ mobilization. No saturation of inositol phosphates accumulation is obtained (up to 10(-2) M ATP). Under the same conditions, ATP rapidly mobilizes intracellular Ca2+ in fura-2 loaded myocytes. The mobilization of intracellular Ca2+ is dose-dependent (maximal at 10(-4) M ATP), and is not affected by addition of EGTA. It is concluded that the receptors mediating the cytosolic increase of Ca2+ are of the P2-purinoceptor subtype. The physiological functions of these receptors are not presently known. 相似文献
6.
Isolated and cultured neonatal cardiac myocytes contract spontaneously and cyclically. The intracellular concentration of free Ca2+ also changes rhythmically in association with the rhythmic contraction of myocytes (Ca2+ oscillation). Both the contraction and Ca2+ oscillatory rhythms are synchronized among myocytes, and intercellular communication via gap junctions has been considered primarily responsible for the synchronization. However, a recent study has demonstrated that intercellular communication via extracellular ATP-purinoceptor signaling is also involved in the intercellular synchronization of intracellular Ca2+ oscillation. In this study, we aim to elucidate whether the concentration of extracellular ATP changes cyclically and contributes to the intercellular synchronization of Ca2+ oscillation among myocytes. In almost all the cultured cardiac myocytes at four days in vitro (4 DIV), intracellular Ca2+ oscillations were synchronized with each other. The simultaneous measurement of the concentration of extracellular ATP and intracellular Ca2+ revealed the extracellular concentration of ATP actually oscillated concurrently with the intracellular Ca2+ oscillation. In addition, power spectrum and cross-correlation analyses suggested that the treatment of cultured cardiac myocytes with suramin, a blocker of P2 purinoceptors, resulted in the asynchronization of Ca2+ oscillatory rhythms among cardiac myocytes. Treatment with suramin also resulted in a significant decrease in the amplitudes of the cyclic changes in both intracellular Ca2+ and extracellular ATP. Taken together, the present study demonstrated the possibility that the concentration of extracellular ATP changes cyclically in association with intracellular Ca2+, contributing to the intercellular synchronization of Ca2+ oscillation among cultured cardiac myocytes. 相似文献
7.
For studying heart pathologies on the cellular level, cultured adult cardiac myocytes represent an important approach. We aimed to explore a novel adult rat ventricular myocyte culture system with minimised dedifferentiation allowing extended experimental manipulation of the cells such as expression of exogenous proteins. Various culture conditions were investigated including medium supplement, substrate coating and electrical pacing for one week. Adult myocytes were probed for (i) viability, (ii) morphology, (iii) frequency dependence of contractions, (iv) Ca(2+) transients, and (v) their tolerance towards adenovirus-mediated expression of the Ca(2+) sensor "inverse pericam". Conventionally, in either serum supplemented or serum-free medium, myocytes dedifferentiated into flat cells within 3 days or cell physiology and morphology were impaired, respectively. In contrast, myocytes cultured in medium supplemented with an insulin-transferrin-selenite mixture on substrates coated with extracellular matrix proteins showed an increased cell attachment and a conserved cross-striation. Moreover, these myocytes displayed optimised preservation of their contractile behaviour and Ca(2+) signalling even under conditions of continuous electrical pacing. Sustained expression of inverse pericam did not alter myocyte function and allowed long lasting high speed Ca(2+) imaging of electrically driven adult myocytes. Our single-cell model thus provides a new advance for high-content screening of these highly specialised cells. 相似文献
8.
Isolated and cultured neonatal cardiac myocytes contract spontaneously and cyclically. The intracellular concentration of free Ca2+ also changes rhythmically in association with the rhythmic contraction of myocytes (Ca2+ oscillation). Both the contraction and Ca2+ oscillatory rhythms are synchronized among myocytes, and intercellular communication via gap junctions has been considered primarily responsible for the synchronization. However, a recent study has demonstrated that intercellular communication via extracellular ATP‐purinoceptor signaling is also involved in the intercellular synchronization of intracellular Ca2+ oscillation. In this study, we aim to elucidate whether the concentration of extracellular ATP changes cyclically and contributes to the intercellular synchronization of Ca2+ oscillation among myocytes. In almost all the cultured cardiac myocytes at four days in vitro (4 DIV), intracellular Ca2+ oscillations were synchronized with each other. The simultaneous measurement of the concentration of extracellular ATP and intracellular Ca2+ revealed the extracellular concentration of ATP actually oscillated concurrently with the intracellular Ca2+ oscillation. In addition, power spectrum and cross‐correlation analyses suggested that the treatment of cultured cardiac myocytes with suramin, a blocker of P2 purinoceptors, resulted in the asynchronization of Ca2+ oscillatory rhythms among cardiac myocytes. Treatment with suramin also resulted in a significant decrease in the amplitudes of the cyclic changes in both intracellular Ca2+ and extracellular ATP. Taken together, the present study demonstrated the possibility that the concentration of extracellular ATP changes cyclically in association with intracellular Ca2+, contributing to the intercellular synchronization of Ca2+ oscillation among cultured cardiac myocytes. 相似文献
9.
We have shown that cystic fibrosis transmembrane conductance regulator (CFTR) is involved in ATP release from skeletal muscle at low pH. These experiments investigate the signal transduction mechanism linking pH depression to CFTR activation and ATP release, and evaluate whether CFTR is involved in ATP release from contracting muscle. Lactic acid treatment elevated interstitial ATP of buffer-perfused muscle and extracellular ATP of L6 myocytes: this ATP release was abolished by the non-specific CFTR inhibitor, glibenclamide, or the specific CFTR inhibitor, CFTRinh-172, suggesting that CFTR was involved, and by inhibition of lactic acid entry to cells, indicating that intracellular pH depression was required. Muscle contractions significantly elevated interstitial ATP, but CFTRinh-172 abolished the increase. The cAMP/PKA pathway was involved in the signal transduction pathway for CFTR-regulated ATP release from muscle: forskolin increased CFTR phosphorylation and stimulated ATP release from muscle or myocytes; lactic acid increased intracellular cAMP, pCREB and PKA activity, whereas IBMX enhanced ATP release from myocytes. Inhibition of PKA with KT5720 abolished lactic-acid- or contraction-induced ATP release from muscle. Inhibition of either the Na+/H+-exchanger (NHE) with amiloride or the Na+/Ca2+-exchanger (NCX) with SN6 or KB-R7943 abolished lactic-acid- or contraction-induced release of ATP from muscle, suggesting that these exchange proteins may be involved in the activation of CFTR. Our data suggest that CFTR-regulated release contributes to ATP release from contracting muscle in vivo, and that cAMP and PKA are involved in the activation of CFTR during muscle contractions or acidosis; NHE and NCX may be involved in the signal transduction pathway. 相似文献
10.
Study of the mechanisms of hydrogen peroxide and hydroxyl free radical-induced cellular injury and calcium overload in cardiac myocytes. 总被引:8,自引:0,他引:8
R A Josephson H S Silverman E G Lakatta M D Stern J L Zweier 《The Journal of biological chemistry》1991,266(4):2354-2361
There is evidence that myocardial injury, as would occur on post-ischemic reperfusion, may be caused by the generation of oxygen radicals, as well as by the induction of intracellular calcium overload; however, the relationship between these two mechanisms of injury is not known. To test the hypothesis that oxidants and oxygen radicals can cause cardiac myocyte injury and intracellular calcium overload, isolated adult rat ventricular myocytes were exposed to H2O2 (1-10 mM) and Fe3(+)-nitrilotriacetate. EPR measurements confirmed the production of the highly reactive .OH radical by this system. The oxygen radical generating system initially caused a transient augmentation of twitch amplitude in single field stimulated myocytes. This was followed by contractile oscillations occurring during the twitch prior to full cell relaxation, and spontaneous mechanical oscillations occurring between electrically stimulated contractions. Eventually, cells became inexcitable and abruptly underwent contracture. In the presence of lower bathing calcium concentrations, these oxidant-induced alterations were prevented or delayed. However, cells exposed to the radical generating system in the absence of extracellular calcium still eventually underwent contracture but stimulated contractions or mechanical oscillations were not seen. Measurements in single myocytes loaded with the fluorescent probe of intracellular calcium, Indo-1, demonstrated a rise in both systolic and diastolic fluorescence ratio, as well as oscillations and widening of the fluorescence transient, suggestive of cellular calcium loading, following exposure to the radical generating system. Injured myocytes did not take up trypan blue dye. Contractile dysfunction and calcium channel blocker, nitrendipine. NMR measurements of cellular [ATP] demonstrated that these alterations in cellular calcium preceded the depletion of ATP. Subsequent depletion of ATP was accompanied by the appearance of increased concentrations of sugar phosphates indicative of a block in glycolysis and ATP depletion correlated with cellular rigor. Thus, oxygen free radicals can cause cardiac myocyte injury with contractile abnormalities which occur due to myocyte calcium loading. The mechanism of oxidant-induced calcium loading is not due to nonspecific membrane damage, or energy depletion, but rather due to increased calcium influx through voltage gated calcium channels. This early calcium overload state as well as oxidant induced block of glycolysis result in cellular energy depletion and cell death with the induction of contracture. 相似文献
11.
Reduction in adipocyte ATP by lipolytic agents: relation to intracellular free fatty acid accumulation 总被引:8,自引:0,他引:8
Epinephrine, norepinephrine, ACTH, and dibutyryl 3',5'-cyclic AMP reduced adipocyte ATP levels during 60 min incubation; glucose displayed a protective effect. The reduction in adipocyte ATP levels could not be attributed solely to: a direct hormone effect, deficiency in metabolic substrate, activation of adenyl cyclase with ATP consumption, loss of adenine nucleotide from the cell or loss of cells during incubation, lipolytic rate per se, or extracellular accumulation of FFA or glycerol. To determine whether intracellular FFA accumulation was a causative factor, intracellular FFA levels were measured during hormone-stimulated lipolysis. This was accomplished by using sucrose-U-(14)C as a marker for the extracellular space to correct for contamination of cells by extracellular albumin-bound FFA. These experiments showed that the fall in adipocyte ATP correlated with FFA saturation of medium albumin and progressive accumulation of FFA within the adipocyte. Furthermore, the protective effect of glucose noted above was associated with a marked reduction in intracellular FFA as compared to the extracellular FFA pool. On the basis of these studies, combined with those in the literature, it is concluded that in vitro effects of lipolytic agents on adipocyte ATP levels are the net result of imparied ATP synthesis (uncoupled oxidative phosphorylation) in the face of normal or augmented ATP consumption. 相似文献
12.
Cardiac organogenesis and pathogenesis are both characterized by changes in myocyte shape, cytoskeletal architecture, and the extracellular matrix (ECM). However, the mechanisms by which the ECM influences myocyte shape and myofibrillar patterning are unknown. We hypothesized that geometric cues in the ECM align sarcomeres by directing the actin network orientation. To test our hypothesis, we cultured neonatal rat ventricular myocytes on islands of micro-patterned ECM to measure how they remodeled their cytoskeleton in response to extracellular cues. Myocytes spread and assumed the shape of circular and rectangular islands and reorganized their cytoskeletons and myofibrillar arrays with respect to the ECM boundary conditions. Circular myocytes did not assemble predictable actin networks nor organized sarcomere arrays. In contrast, myocytes cultured on rectangular ECM patterns with aspect ratios ranging from 1:1 to 7:1 aligned their sarcomeres in predictable and repeatable patterns based on highly localized focal adhesion complexes. Examination of averaged alpha-actinin images revealed invariant sarcomeric registration irrespective of myocyte aspect ratio. Since the sarcomere sub-units possess a fixed length, this observation indicates that cytoskeleton configuration is length-limited by the extracellular boundary conditions. These results indicate that modification of the extracellular microenvironment induces dynamic reconfiguring of the myocyte shape and intracellular architecture. Furthermore, geometric boundaries such as corners induce localized myofibrillar anisotropy that becomes global as the myocyte aspect ratio increases. 相似文献
13.
In preliminary experiments it was established that the hypertrophic and hyperplastic responses of neonatal cardiac myocytes in culture were associated with enhanced expression of IGF-1 and IGF-1 receptors in these cells. Therefore, to determine the role of IGF-1 receptors on myocyte growth, cells were exposed to antisense oligodeoxynucleotides to IGF-1 receptor mRNA and the effects of this intervention on DNA synthesis, nuclear mitotic division, and changes in the number of myocytes were measured. Moreover, the influence of this procedure on ANF induction and myocyte cell volume was examined. Inhibition of the formation of IGF-1 receptors on myocytes suppressed DNA replication, mitosis, and cell proliferation. In contrast, the antisense treatment did not alter the expression of ANF in myocytes or cellular hypertrophy. Finally, IGF-1 stimulated DNA synthesis in myocytes cultured in serum-free medium, without inducing cellular hypertrophy. In conclusion, ligand activation of IGF-1 receptors on myocytes appears to be coupled with cell proliferation, whereas myocyte cellular hypertrophy seems to be independent from this effector pathway. 相似文献
14.
Embryonic Xenopus myocytes generate spontaneous calcium (Ca(2+)) transients during differentiation in culture. Suppression of these transients disrupts myofibril organization and the formation of sarcomeres through an identified signal transduction cascade. Since transients often occur during myocyte polarization and migration in culture, we hypothesized they might play additional roles in vivo during tissue formation. We have tested this hypothesis by examining Ca(2+) dynamics in the intact Xenopus paraxial mesoderm as it differentiates into the mature myotome. We find that Ca(2+) transients occur in cells of the developing myotome with characteristics remarkably similar to those in cultured myocytes. Transients produced within the myotome are correlated with somitogenesis as well as myocyte maturation. Since transients arise from intracellular stores in cultured myocytes, we examined the functional distribution of both IP(3) and ryanodine receptors in the intact myotome by eliciting Ca(2+) elevations in response to photorelease of caged IP(3) and superfusion of caffeine, respectively. As in culture, transients in vivo depend on Ca(2+) release from ryanodine receptor (RyR) stores, and blocking RyR during development interferes with somite maturation. 相似文献
15.
Accumulation and salvage of adenosine and inosine by isolated mature cardiac myocytes 总被引:1,自引:0,他引:1
Reoxygenation of ischaemic, energy-depleted heart does not result in sufficiently rapid regeneration of normal adenine nucleotide concentrations for preservation of cardiac function and structure. Salvage of nucleoside as a mechanism for restoration of ATP in the post-ischaemic myocardium is limited by efflux of adenosine during ischaemia. Isolated cardiac myocytes have been used to establish the kinetics of uptake and salvage of adenosine and inosine, measuring the distribution of radioactive nucleoside incorporated into ATP, ADP and AMP. Maximum rates of catalysis of reactions on the salvage pathway, and of enzymes competing for substrates on the pathway, have been established in myocyte extracts. Myocytes have little capacity to salvage or catabolise inosine. Enzyme measurements indicate that salvage of adenosine should proceed at 7-8-times the rate exhibited by intact myocytes dependent upon extracellular adenosine as substrate. The data indicate that the rate of transport of adenosine is not determined by its metabolic utilization, but is the rate-limiting step in the salvage of adenosine. 相似文献
16.
Terje K. Steigen Truls Myrmel Kirsti Forsdahl Terje S. Larsen 《Molecular and cellular biochemistry》1992,116(1-2):47-52
Myocardial cell vulnerability to phospholipase C (PC-PLC) attack was investigated in three different preparations of rat myocardial cells: triacylglycerol (TG)-loaded, hypothermic/rewarmed and energy depleted myocytes. The attack by PC-PLC was evaluated as PC-PLC induced glycerol output due to the combined action of phospholipase C and intracellular lipases. PC-PLC induced glycerol output was significantly higher (p < 0.05) in all three myocyte preparations, compared to their respective controls. Cell morphology (% rod shaped myocytes) of TG-loaded or hypothermic/rewarmed myocytes was not different from their controls, whereas energy depleted myocytes almost exclusively were rounded up, due to hypercontraction of the myofilaments. Hypothermic/rewarmed and energy depleted myocytes showed a significantly higher release of lactate dehydrogenase (LDH), compared to their controls although the difference was much more pronounced in the latter. Finally, the cellular contents of ATP were maintained both in TG-loaded and hypothermic rewarmed myocytes, while energy depleted myocytes contained only about 25% of the normal ATP level. These results demonstrate that attack from exogenously added phospholipases can occur, not only in seriously damaged cardiac myocytes, but in myocytes with a more subtle damage as well. (Mol Cell Biochem 116: 47–52, 1992) 相似文献
17.
L L Hilenski X H Ma N Vinson L Terracio T K Borg 《Cell motility and the cytoskeleton》1992,21(2):87-100
The influence of the extracellular matrix (ECM) on cell behavior, myofibrillogenesis and cytoarchitecture was investigated in neonatal rat cardiac myocytes in vitro. Cell behavior was examined by analyzing cell spreading on different ECM components under a variety of experimental conditions. Area measurements were made on digitized images of cells grown for various time intervals on fibronectin (FN), laminin (LN), collagens I and III (C I+III), plastic, and bovine serum albumin (BSA). The amount of spreading was varied on the different matrices and was maximal on FN greater than LN greater than C I+III greater than plastic greater than BSA. Addition of anti-beta 1 integrin antibodies to myocytes cultured on FN, LN and C I+III blocked spreading outward on the substrates and altered normal myofibrillogenesis, especially on LN. Concomitantly, the integrin antibodies induced the formation of giant pseudopodial processes which protruded upward from the substrates. These pseudopods contained actin polygonal networks which exhibited a regular geometrical configuration. Effects of the ECM on cytoarchitecture was examined by analyzing the temporal and spatial patterns of fluorescence and immunogold labeling of cytoskeletal and integrin proteins as myocytes spread in culture. The first indication of sarcomeric patterns was the appearance at 4 hours of striations formed by lateral alignment of alpha-actinin aggregates into Z bands. At later times, vinculin at 8 hours and beta 1 integrin at 22 hours became co-localized with alpha-actinin at the Z bands and focal adhesions. These data indicate that ECM components influence myocyte spreading and that myofibril assembly and/or stability is associated with ECM-integrin-cytoskeleton associations. 相似文献
18.
Heterokaryon analysis of muscle differentiation: regulation of the postmitotic state 总被引:10,自引:5,他引:5
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MM14 mouse myoblasts withdraw irreversibly from the cell cycle and become postmitotic within a few hours of being deprived of fibroblast growth factor (Clegg, C. H., T. A. Linkhart, B. B. Olwin, and S. D. Hauschka, 1987, J. Cell Biol., 105:949-956). To examine the mechanisms that may regulate this developmental state of skeletal muscle, we tested the mitogen responsiveness of various cell types after their polyethylene glycol-mediated fusion with post-mitotic myocytes. Heterokaryons containing myocytes and quiescent nonmyogenic cells such as 3T3, L cell, and a differentiation-defective myoblast line (DD-1) responded to mitogen-rich medium by initiating DNA synthesis. Myonuclei replicated DNA and reexpressed thymidine kinase. In contrast, (myocyte x G1 myoblast) heterokaryons failed to replicate DNA in mitogen-rich medium and became postmitotic. This included cells with a nuclear ratio of three myoblasts to one myocyte. Proliferation dominance in (myocyte x 3T3 cell) and (myocyte x DD-1) heterokaryons was conditionally regulated by the timing of mitogen treatment; such cells became postmitotic when mitogen exposure was delayed for as little as 6 h after cell fusion. In addition, (myocyte x DD-1) heterokaryons expressed a muscle-specific trait and lost epidermal growth factor receptors when they became postmitotic. These results demonstrate that DNA synthesis is not irreversibly blocked in skeletal muscle; myonuclei readily express proliferation-related functions when provided with a mitogenic signal. Rather, myocyte-specific repression of DNA synthesis in heterokaryons argues that the postmitotic state of skeletal muscle is regulated by diffusible factors that inhibit processes of cellular mitogenesis. 相似文献
19.
Release of enzymes from human fibroblasts, as a result of either ATP depletion caused by metabolic inhibitors or a direct cell membrane damage, was increased in the absence of extracellular Ca2+. The lack of extracellular Mg2+ resulted only in a small nonsignificant increase of enzyme release during ATP depletion. However, in a Ca2+-free medium the enzyme release was very much enhanced during ATP depletion if Mg2+ was absent concomitantly. The data show that extracellular calcium protects the cells from cell damage, and that the presence of extracellular magnesium in a calcium-free medium protects the cell against energy-dependent cell damage. 相似文献
20.
Chemical hypoxia triggers apoptosis of cultured neonatal rat cardiac myocytes: Modulation by calcium-regulated proteases and protein kinases 总被引:10,自引:0,他引:10
Chen Shu Jen Bradley Michael E. Lee Te Chung 《Molecular and cellular biochemistry》1998,178(1-2):141-149
Myocardial infarctions and stroke arise primarily as a result of hypoxia/ischemia-induced cell injury. However, the molecular mechanism of cardiac cell death due to hypoxia has not been elucidated. We showed here that chemical hypoxia induced by 1 mM azide triggered apoptosis of isolated neonatal rat ventricular cardiac myocytes but had no effect on cardiac fibroblasts. The azide-induced cardiomyocyte apoptosis could be characterized by a reversible initiation phase (0-6 h after azide exposure) during which cytosolic ATP levels remained little affected. This was followed by an irreversible execution phase (12-18 h) exhibiting prominent internucleosomal DNA fragmentation, cell membrane leakage, mitochondrial dysfunction, and increased calpain messenger RNA. Blocking extracellular calcium influx or intracellular calcium release was each effective in suppressing myocyte apoptosis. Cell death was also found to be mediated by calcium sensitive signal transduction events based on the use of specific antagonists. Consistent with the induction of calpain expression during apoptosis, blocking de novo protein synthesis and calpain activity inhibited cell death. These regulatory features coupled with the ease of the cell system suggest that the myocyte apoptosis model described here should be useful in the study of events leading to the demise of the myocardium. 相似文献