Extracellular matrix mimicking scaffold promotes osteogenic stem cell differentiation: A new approach in osteoporosis research.

BACKGROUND: Osteoporosis is a common metabolic disease, with mesenchymal stem cells discussed to play an important role in its pathomechanism. For in vitro osteoporosis studies, selection of adequate culture conditions is mandatory so as to preserve cell properties as far as possible. A suitable cell culture surface would ideally provide reproducible experimental conditions by resembling those in-vivo. OBJECTIVE: Generating an improved growth surface for osteogenic differentiation of human bone marrow derived mesenchymal stem cells (hBMSCs). METHODS: We modified electrospun gelatine meshes with hydroxyapatite nanopowder. The potential beneficial impact of the ensuing culture conditions were evaluated by cultivating and comparing the growth of cells from osteoporotic and non-osteoporotic donors on either hydroxyapatite-gelatine (HA) meshes, pure gelatine meshes, or 2D standard tissue culture surfaces. RESULTS: After 21 days of differentiation, cells grown on pure or HA-gelatine meshes showed significantly higher mineralization levels compared to cells cultured in standard conditions. The amount of mineralization varied considerably in hBMSC cultures of individual patients but showed no significant difference between stem cells obtained from osteoporotic or non-osteoporotic donors. CONCLUSIONS: Overall, these results indicate that the use of HA-gelatine meshes as growth surfaces may serve as a valuable tool for cultivation and differentiation of mesenchymal stem cells along the osteogenic lineage, facilitating future research on osteoporosis and related issues.

Death of p53-defective cells triggered by forced mitotic entry in the presence of DNA damage is not uniquely dependent on Caspase-2 or the PIDDosome.

Much effort has been put in the discovery of ways to selectively kill p53-deficient tumor cells and targeting cell cycle checkpoint pathways has revealed promising candidates. Studies in zebrafish and human cell lines suggested that the DNA damage response kinase, checkpoint kinase 1 (Chk1), not only regulates onset of mitosis but also cell death in response to DNA damage in the absence of p53. This effect reportedly relies on ataxia telangiectasia mutated (ATM)-dependent and PIDDosome-mediated activation of Caspase-2. However, we show that genetic ablation of PIDDosome components in mice does not affect cell death in response to γ-irradiation. Furthermore, Chk1 inhibition largely failed to sensitize normal and malignant cells from p53(-/-) mice toward DNA damaging agents, and p53 status did not affect the death-inducing activity of DNA damage after Chk1 inhibition in human cancer cells. These observations argue against cross-species conservation of a Chk1-controlled cell survival pathway demanding further investigation of the molecular machinery responsible for cell death elicited by forced mitotic entry in the presence of DNA damage in different cell types and model organisms.

Loss of PIDD limits NF-κB activation and cytokine production but not cell survival or transformation after DNA damage.

Activation of NF-κB (nuclear factor of kappa light chain gene enhancer in B cells) in response to DNA damage is considered to contribute to repair of genetic lesions, increased cell survival and cytokine release. The molecular mechanisms orchestrating this cytoplasmic event involve core components of the nuclear DNA damage response machinery, including ATM-kinase (ataxia telangiectasia mutated kinase) and PARP-1 (poly (ADP-ribose) polymerase 1). The physiological consequences of defective NF-κB activation in this context, however, remain poorly investigated. Here we report on the role of the 'p53-induced protein with a death domain', PIDD, which appears rate limiting in this process, as is PARP-1. Despite impaired NF-κB activation, DNA damage did not increase cell death or reduce clonal survival of various cell types lacking PIDD, such as mouse embryonic fibroblasts or stem and progenitor cells of the hematopoietic system. Furthermore, lymphomagenesis induced by γ-irradiation (IR) was unaffected by deficiency for PIDD or PARP-1, indicating that loss of DNA damage-triggered NF-κB signalling does not affect IR-driven tumorigenesis. However, loss of either gene compromised cytokine release after acute IR injury. Hence, we propose that NF-κB's most notable function after DNA damage in primary cells is related to the release of cytokines, thereby contributing to sterile inflammation.

PIDDosome-independent tumor suppression by Caspase-2.

The PIDDosome, a multiprotein complex constituted of the 'p53-induced protein with a death domain (PIDD), 'receptor-interacting protein (RIP)-associated ICH-1/CED-3 homologous protein with a death domain' (RAIDD) and pro-Caspase-2 has been defined as an activating platform for this apoptosis-related protease. PIDD has been implicated in p53-mediated cell death in response to DNA damage but also in DNA repair and nuclear factor kappa-light-chain enhancer (NF-κB) activation upon genotoxic stress, together with RIP-1 kinase and Nemo/IKKγ. As all these cellular responses are critical for tumor suppression and deregulated expression of individual PIDDosome components has been noted in human cancer, we investigated their role in oncogenesis induced by DNA damage or oncogenic stress in gene-ablated mice. We observed that Pidd or Caspase-2 failed to suppress lymphoma formation triggered by γ-irradiation or 3-methylcholanthrene-driven fibrosarcoma development. In contrast, Caspase-2 showed tumor suppressive capacity in response to aberrant c-Myc expression, which did not rely on PIDD, the BH3-only protein Bid (BH3 interacting domain death agonist) or the death receptor ligand Trail (TNF-related apoptosis-inducing ligand), but associated with reduced rates of p53 loss and increased extranodal dissemination of tumor cells. In contrast, Pidd deficiency associated with abnormal M-phase progression and delayed disease onset, indicating that both proteins are differentially engaged upon oncogenic stress triggered by c-Myc, leading to opposing effects on tumor-free survival.

BH3-only protein Bmf mediates apoptosis upon inhibition of CAP-dependent protein synthesis.

Tight transcriptional regulation, alternative splicing and/or post-translational modifications of BH3-only proteins fine-tune their proapoptotic function. In this study, we characterize the gene locus of the BH3-only protein Bmf (Bcl-2-modifying factor) and describe the generation of two major isoforms from a common transcript in which initiation of protein synthesis involves leucine-coding CUG. Bmf(CUG) and the originally described isoform, Bmf-short, display comparable binding affinities to prosurvival Bcl-2 family members, localize preferentially to the outer mitochondrial membrane and induce rapid Bcl-2-blockable apoptosis. Notably, endogenous Bmf expression is induced on forms of cell stress known to cause repression of the CAP-dependent translation machinery such as serum deprivation, hypoxia, inhibition of the PI3K/AKT pathway or mTOR, as well as direct pharmacological inhibition of the eukaryotic translation initiation factor eIF-4E. Knock down or deletion of Bmf reduces apoptosis under some of these conditions, demonstrating that Bmf can act as a sentinel for stress-impaired CAP-dependent protein translation machinery.

Caspase-2: killer, savior and safeguard--emerging versatile roles for an ill-defined caspase.

Despite the early discovery of caspase-2, its physiological function has long remained an enigma. A number of recent publications now suggest not just one, but multiple functions, including roles in apoptosis, DNA repair and tumor suppression. How can one enzyme have so many talents? Considering the diversity of interaction partners and the specific mode of pro-apoptotic action proposed in these studies, caspase-2 might in fact represent a primordial protease serving numerous pathways, established before the advent of a more elaborate functionally diversified caspases system.

The enigma of caspase-2: the laymen's view.

Proteolysis of cellular substrates by caspases (cysteine-dependent aspartate-specific proteases) is one of the hallmarks of apoptotic cell death. Although the activation of apoptotic caspases is considered a 'late-stage' event in apoptosis signaling, past the commitment stage, one caspase family member, caspase-2, splits the cell death community into half - those searching for evidence of an apical initiator function of this molecule and those considering it as an amplifier of the apoptotic caspase cascade, at best, if relevant for apoptosis at all. This review screens past and present biochemical as well as genetic evidence for caspase-2 function in cell death signaling and beyond.

Volumetric response of vertebrate hepatocytes challenged by osmotic gradients: a theoretical approach.

In this study we use a theoretical approach to study the volumetric response of goldfish hepatocytes challenged by osmotic gradients and compared it with that of hepatocytes from another teleost (the trout) and a mammal (the rat). Particular focus was given to the multiple non-linear interactions of transport systems enabling hypotonically challenged cells to trigger a compensatory response known as volume regulatory decrease or RVD. For this purpose we employed a mathematical model which describes the rates of change of the intracellular concentrations of main diffusible ions, of the cell volume, and of the membrane potential. The model was fitted to experimental data on the kinetics of volume change of hepatocytes challenged by anisotonic media. In trout and rat hepatocytes, experimental results had shown that hypotonic cell swelling was followed by RVD, whereas goldfish cells swelled with no concomitant RVD (M.V. Espelt et al., 2003, J. Exp. Biol. 206, 513-522). A comparison between data predicted by the model and that obtained experimentally suggests that in trout and rat hepatocytes hypotonicity activates a sensor element and this, in turn, activates an otherwise silent efflux of KCl - whose kinetics could be successfully predicted - thereby leading to volume down-regulation. In contrast, with regard to the absence of RVD in goldfish hepatocytes the model proposed suggests that either a sensor element triggering RVD is absent or that the effector mechanism (the loss of KCl) remains inactive under the conditions employed. In line with this, we recently found that extracellular nucleotides may be required to induce RVD in these cells, indicating that our model could indeed lead to useful predictions.

Potassium transmembrane fluxes in anoxic hepatocytes from goldfish (Carassius auratus L.).

Despite the fact that anoxic goldfish hepatocytes can maintain the transmembrane gradients of Na(+), H(+) and Ca(2+), cyanide (CN) intoxication leads to a rapid breakdown of K(+) homeostasis. In this study, [(86)Rb(+)] K(+) fluxes across the plasma membrane of goldfish hepatocytes were studied in order to identify the possible causes of this imbalance. Four minutes of cyanide incubation induced an acute and stable 61% decrease of K(+) influx (mostly driven by Na,K-ATPase activity), whereas K(+) efflux increased by 24.3%, this imbalance yielding a net K(+) efflux of 0.279+/-0.024 nmol 10(-6) cells(-1) min(-1). This uncoupling was not observed when glycolytic ATP production was inhibited with iodoacetic acid. Although the CN-induced decrease of K(+) influx was fully reversible upon washout of the inhibitor, it could not be prevented by any of the following treatments: (1) addition of 2% bovine serum albumin, which binds extracellular fatty acids known to activate specific K(+) channels; (2) addition of ascorbate, which acts as a radical scavenger; (3) inclusion of 5 mM glucose as an extracellular carbon source; and (4) removal of medium oxygen (obtained by nitrogen bubbling). Regarding the elevation of K(+) efflux in the presence of CN, neither ATP-dependent K(+) channels nor the KCl cotransporter appeared to be activated, whereas BaCl(2), an inhibitor of voltage-gated K(+) channels, decreased K(+) efflux of CN-intoxicated cells to control levels. In summary, these results indicate that, in goldfish hepatocytes, the CN-induced K(+) imbalance results from acute Na,K-ATPase inhibition together with the activation of voltage-dependent K(+) channels, the latter probably resulting from transient membrane depolarization.

Effects of extracellular nucleotides and their hydrolysis products on regulatory volume decrease of trout hepatocytes.

In trout hepatocytes, hypotonic swelling is followed by a compensatory shrinkage called regulatory volume decrease (RVD). It has been postulated that extracellular ATP and other nucleotides may interact with type 2 receptors (P(2)) to modulate this response. In addition, specific ectoenzymes hydrolyze ATP sequentially down to adenosine, which may bind to type 1 receptors (P(1)) and also influence RVD. Accordingly, in this study, we assessed the role of extracellular nucleoside 5'-tri- and diphosphates and of adenosine on RVD of trout hepatocytes. The extent of RVD after 40 min of maximum swelling was denoted as RVD(40), whereas the initial rate of RVD was called v(RVD). In the presence of hypotonic medium (60% of isotonic), hepatocytes swelled 1.6 times followed by v(RVD) of 1.7 min(-1) and RVD(40) of 60.2%. ATP, UTP, UDP, or ATPgammaS (P(2) agonists; 5 microM) increased v(RVD) 1.5-2 times, whereas no changes were observed in the values of RVD(40). Addition of 100 microM suramin or cibacron blue (P(2) antagonists) to the hypotonic medium produced no effect on v(RVD) but a 53-58% inhibition of RVD(40). Incubation of hepatocytes in the presence of either 5 microM [gamma-(32)P]ATP or [alpha-(32)P]ATP induced the extracellular release of [gamma-(32)P]P(i) (0.21 nmol.10(-6) cells(-1).min(-1)) and [alpha-(32)P]P(i) ( approximately 8 x 10(-3) nmol.10(-6) cells(-1).min(-1)), suggesting the presence of ectoenzymes capable of fully dephosphorylating ATP. Concerning the effect of P(1) activation on RVD, 5 microM adenosine, both in the presence and absence of 100 microM S-(4-nitrobenzil)-6-tioinosine (a blocker of adenosine uptake), decreased RVD(40) by 37-44%, whereas 8-phenyl theophylline, a P(1) antagonist, increased RVD(40) by 15%. Overall, results indicate that ATP, UTP, and UDP, acting via P(2), are important factors promoting RVD of trout hepatocytes, whereas adenosine binding to P(1) inhibits this process.

Volumetric and ionic responses of goldfish hepatocytes to anisotonic exposure and energetic limitation.

The relationship between cell volume and K(+) transmembrane fluxes of goldfish (Carassius auratus) hepatocytes exposed to anisotonic conditions or energetic limitation was studied and compared with the response of hepatocytes from trout (Oncorhynchus mykiss) and rat (Rattus rattus). Cell volume was studied by video- and fluorescence microscopy, while K(+) fluxes were assessed by measuring unidirectional (86)Rb(+) fluxes. In trout and rat hepatocytes, hyposmotic (180 mosmoll(-1)) exposure at pH 7.45 caused cell swelling followed by a regulatory volume decrease (RVD), a response reported to be mediated by net efflux of KCl and osmotically obliged water. By contrast, goldfish hepatocytes swelled but showed no RVD under these conditions. Although in goldfish hepatocytes a net ((86)Rb(+))K(+) efflux could be activated by N-ethylmaleimide, this flux was not, or only partially, activated by hyposmotic swelling (120-180 mosmoll(-1)). Blockage of glycolysis by iodoacetic acid (IAA) did not alter cell volume in goldfish hepatocytes, whereas in the presence of cyanide (CN(-)), an inhibitor of oxidative phosphorylation, or CN(-) plus IAA (CN(-)+IAA), cell volume decreased by 3-7%. Although in goldfish hepatocytes, energetic limitation had no effect on ((86)Rb(+))K(+) efflux, ((86)Rb(+))K(+) influx decreased by 57-66% in the presence of CN(-) and CN(-)+IAA but was not significantly altered by IAA alone. Intracellular K(+) loss after 20 min of exposure to CN(-) and CN(-)+IAA amounted to only 3% of the total intracellular K(+). Collectively, these observations suggest that goldfish hepatocytes, unlike hepatocytes of anoxia-intolerant species, avoid a decoupling of transmembrane K(+) fluxes in response to an osmotic challenge. This may underlie both the inability of swollen cells to undergo RVD but also the capability of anoxic cells to maintain intracellular K(+) concentrations that are almost unaltered, thereby prolonging cell survival.

Identification of two distinct E-NTPDases in liver of goldfish (Carassius auratus L.).

We have recently reported the existence of ATPase activity capable of hydrolyzing extracellular ATP and localized at the external cell membrane of goldfish hepatocytes [Am. J. Physiol. (1998) 274 R1031]. In the present study, we investigated whether one or more enzymes of the ATP diphosphohydrolase family (called E-NTPDases) are responsible for the hydrolysis of extracellular ATP and other nucleotides. Using soluble extracts from goldfish liver, enzyme activity was detected in the presence of ATP (32.1+/-4.0 nmol Pi liberated mg protein(-1) min(-1)), ADP (20.7+/-3.3 nmol Pi liberated mg protein(-1) min(-1)) and UTP (20.7+/-1.2 nmol Pi liberated mg protein(-1) min(-1)). In line with the presence of this hydrolytic activity, liver samples separated by non-denaturing gel electrophoresis and subsequently exposed to either ATP, ADP or UTP yielded a single band with enzyme activity and similar electrophoretic mobility. Subsequent SDS-PAGE electrophoresis of the active bands resulted in the appearance of two protein bands with molecular masses of 70 and 64 kDa. Immunoblotting of soluble extracts and microsomes obtained from goldfish liver, using a monoclonal antibody against CD39 (a well-known E-NTPDase), detected a single 97-kDa protein. The enzyme activity measured in solution and in native gels, together with structural information from denaturing gels plus immunoblots, points to the existence, in goldfish liver, of at least two different E-NTPDases.

Importance of glycolysis for the energetics of anoxia-tolerant and anoxia-intolerant teleost hepatocytes.

The importance of glycolysis, as an ATP-producing and substrate-providing pathway, was studied in anoxia-tolerant (goldfish) and anoxia-intolerant (trout) hepatocytes. Inhibition of glycolysis with iodoacetic acid (IAA) left aerobic ATP production largely unaffected in hepatocytes from both species but caused a significant decrease of ATP contents in the goldfish cells. Ouabain-sensitive oxygen consumption (osVo2), an estimate of mitochondrial ATP production coupled to ATP consumption by the Na(+) pump, was significantly reduced in IAA-treated goldfish hepatocytes, whereas it was unaltered in trout hepatocytes. Partial reduction of mitochondrial respiration, achieved by titration with cyanide (CN), strongly stimulated glycolytic flux but did not affect ATP contents of hepatocytes from both species. Under these conditions, osVo2 became undetectable. Rb(+)-uptake rates, providing a direct estimate of Na(+)-pump activity, were in good agreement with estimates derived from osVo2 in IAA-treated cells, showing a decrease in goldfish and no change in trout. However, they indicated persistent Na(+)-pump activity despite the lack of osVo2 in CN-treated cells. Overall, these data indicate that in goldfish hepatocytes Na(+)-pump activity is more dependent on glycolytic ATP production as compared to trout hepatocytes. Protein synthesis of goldfish hepatocytes was inhibited in IAA- and CN-treated cells, possibly reflecting the hierarchical organization of energy metabolism. In trout hepatocytes, protein synthesis could be sustained at control levels, given that energetic substrate provision was not limited.

Hierarchies of ATP-consuming processes: direct compared with indirect measurements, and comparative aspects.

The original aim of the present study was to deal with two problems that had emerged from a study on hierarchies of ATP-consuming processes in cells [Buttgereit and Brand (1995) Biochem. J. 312, 163-167]. Firstly, we wanted to find out whether the results of that study had been influenced by the method used for the determination of process activity and, secondly, we wondered whether and to what extent the structure of the hierarchy established for cell suspensions under energy-limiting conditions might depend on the type of cell or on the lifestyle, ecology and phylogenetic status of the species from which the cells were derived. We confined our study to the two most prominent ATP consumers of cells: protein synthesis and the Na(+)/K(+)-ATPase, measuring their activity directly by [3H]leucine incorporation and Rb(+)-flux respectively. We found large differences in the sensitivity of protein synthesis to energy limitation between hepatocytes from an anoxia-tolerant fish species and an anoxia-sensitive fish species (goldfish and rainbow trout respectively). On the other hand, Na(+)/K(+)-ATPase activity was hardly affected by energy limitation in the hepatocytes from both fish species. We also studied the response of a human hepatoma cell line, HepG2, to energy limitation and found both protein synthesis and Na(+)/K(+)-ATPase activity to be equally sensitive to energy limitation, but more sensitive than the Na(+)/K(+)-ATPase of the two fish species. A comparison of the indirect and direct methods for measuring protein synthesis revealed the rate of oxygen consumption to be functionally related to the concentration of cycloheximide, the inhibitor used. It was found that at 15 mM cycloheximide [three orders of magnitude higher than the concentration at which the incorporation of free amino acids (FAA) into protein is inhibited] total oxygen consumption was suppressed by 71-75%, whereas the measured rate of [3H]leucine incorporation into protein suggested that the cycloheximide-sensitive fraction should have amounted to not more than approx. 10% of the total oxygen consumption. On the other hand, the amount of oxygen consumption suppressed with the high concentration of cycloheximide corresponded almost exactly to the increase in oxygen consumption of cells incubated in an FAA-enriched medium compared with cells incubated in a standard, FAA-free medium. Our major conclusions are, firstly, that high concentrations of cycloheximide disrupt cellular metabolism, bringing to a standstill all those processes that can be stimulated by incubating starved cells in an FAA-enriched medium, secondly, that the attempt to estimate the metabolic cost of protein synthesis by inhibiting oxygen consumption with cycloheximide leads to spurious results, and, thirdly, that the structure of a 'hierarchy' of ATP-consumers may reflect the lifestyle and physiology of the species studied.

Metabolic responses to epinephrine stimulation in goldfish hepatocytes: evidence for the presence of alpha-adrenoceptors.

The effect of epinephrine on various aspects of cellular metabolism was studied in hepatocytes from the goldfish Carassius auratus. Epinephrine increased cytosolic free calcium ([Ca2+](i)) from a baseline value of 108 +/- 22 nM to a peak value of 577 +/- 127 nM in suspensions of hepatocytes. Responses of single cells ranged from a single spike (66% of hepatocytes) to variable oscillatory patterns (34%). The increase in [Ca(2+)](i) was independent of the presence of extracellular Ca2+ and was prevented by the alpha-adrenergic antagonist phentolamine. Cellular glucose release induced by epinephrine (1.7- to 3.2-fold) was significantly reduced in Ca2+-depleted cells and in the presence of phentolamine, providing evidence for the co-occurrence of alpha-adrenoceptors and a Ca2+-independent, presumably beta-adrenergic, system in these cells. Furthermore, epinephrine stimulated oxygen consumption in a Ca2+-dependent manner, which was not due to stimulated Na(+) pump activity. An increased rate of acid secretion of 50%, evoked by epinephrine, appears to be mediated by enhanced Na(+)/H(+) exchange but did not result in intracellular alkalization.

Regulation of intracellular pH in anoxia-tolerant and anoxia-intolerant teleost hepatocytes.

Mechanisms of intracellular pH (pHi) regulation were investigated in anoxia-tolerant hepatocytes from goldfish Carassius auratus, and compared to the situation in the anoxia-intolerant hepatocytes from trout Oncorhynchus mykiss. Under normoxic conditions, the pHi of goldfish hepatocytes was regulated by a Na(+)/H(+) exchanger and a Na(+)-independent Cl(-)/HCO(3)(-) exchanger, the latter being activated only after acidification of the cells. Mechanisms of acid secretion appear to be fuelled, at least in part, by lactate formation under fully aerobic conditions, as inhibition of glycolysis caused a drastic reduction of steady state proton release. In trout hepatocytes both a Na(+)/H(+) exchanger and a Cl(-)/HCO(3)(-) exchanger were found to be tonically active, as described previously. During chemical anoxia a constant pHi was maintained in goldfish hepatocytes, whereas it was reversibly reduced by 0.3 units in the trout cells. Under these conditions a reversible increase in the rate of acid secretion was induced in the cells from both species. In the goldfish cells this was based on a SITS-sensitive transporter, possibly involving export of lactate, with no contribution from Na(+)/H(+) exchange. By contrast, in hepatocytes from trout, CN-induced acid secretion was dominated by the activity of the Na(+)/H(+) exchanger. Brief exposure to extracellular acidosis had no dramatic effects on the energetics of hepatocytes from either species.

Action of adenosine on energetics, protein synthesis and K(+) homeostasis in teleost hepatocytes.

In a comparative study, we analysed the effects of adenosine on the energetics, protein synthesis and K(+ )homeostasis of hepatocytes from the anoxia-tolerant goldfish Carassius auratus and the anoxia-intolerant trout Oncorhynchus mykiss. The rate of oxygen consumption did not respond immediately to the addition of adenosine to the cells from either species, but showed a significant decrease in trout hepatocytes after 30 min. The anaerobic rate of lactate formation was not significantly affected by adenosine in goldfish hepatocytes, but was increased in trout cells. We also studied the effects of adenosine on the two most prominent ATP consumers in these cells, protein synthesis and Na(+)/K(+)-ATPase activity. Under aerobic conditions, adenosine inhibited protein synthesis of hepatocytes from goldfish by 51% and of hepatocytes from trout by 32%. During anoxia, the rate of protein synthesis decreased by approximately 50% in goldfish hepatocytes and by 90% in trout hepatocytes, and this decrease was not altered by the presence of adenosine. Adenosine inhibited normoxic Na(+)/K(+)-ATPase activity and K(+ )efflux by 20-35% in the cells of both species. An investigation into the mechanism underlying the inhibition of protein synthesis by adenosine indicated that, in the goldfish cells, adenosine acts via a membrane receptor-mediated pathway, i.e. the effect of adenosine was abolished by applying the A1 receptor antagonist 8-phenyltheophylline. In the trout, however, the uptake of adenosine into hepatocytes seems to be required for an effect on protein synthesis. [Ca(2+)](i) does not seem to be involved in the inhibition of protein synthesis by adenosine.

Oxygen-dependent energetics of anoxia-tolerant and anoxia-intolerant hepatocytes.

The oxygen-dependence of cellular energetics was investigated in hepatocytes from goldfish Carassius auratus (anoxia-tolerant) and rainbow trout Oncorhynchus mykiss (anoxia-intolerant). In goldfish hepatocytes, an approximately 50 % reduction in the rate of oxygen consumption was observed in response to both acute and prolonged hypoxia, the latter treatment shifting the threshold for this reduction to a higher oxygen level. A concomitant increase in the rate of lactate production did not compensate for the decreased aerobic ATP supply, resulting in an overall metabolic depression of 26 % during acute hypoxia and of 42 % during prolonged hypoxia. Trout hepatocytes showed a similar suppression of cellular respiration after prolonged hypoxia but were unresponsive to acute hypoxia. Similarly, the rate of lactate production was unaltered during acute hypoxia but was increased during prolonged hypoxia, metabolic depression amounting to 7 % during acute hypoxia and 30 % during prolonged hypoxia. In both species, the affinity of hepatocytes for oxygen decreased during hypoxia, but this alteration was not sufficient in absolute terms to account for the observed decrease in aerobic ATP supply. Protein synthesis was suppressed in both cell types under hypoxia, whereas Na(+)/K(+)-ATPase activity decreased in trout but not in goldfish hepatocytes, emphasising the importance of membrane function in these cells during conditions of limited energy supply.

Energetics of trout hepatocytes during A23187-induced disruption of Ca2+ homeostasis.

The impact of an increase of intracellular Ca2+ i on the energy metabolism of trout hepatocytes was assessed by applying the Ca2+ ionophore A23187 and studying the consequences of the ensuing elevation of Ca2+ i on various metabolic parameters. After application of A23187 no loss of viability occurred for 2 h, but glutathione content decreased by 46%. A concomitant decrease of [ATP] as well as of Na,K-ATPase activity by over 50% could be prevented by incubating the cells in a Ca2+-free medium. Upon addition of the ionophore cellular oxygen consumption more than doubled in a strictly Ca2+-dependent manner, with half of this increase being sensitive to ruthenium red, an inhibitor of the mitochondrial Ca2+ uniporter. This increase in oxygen consumption was transient in nature and at its peak it was similar in magnitude to that induced by 2,4-dinitrophenol. Similarly, oxygen consumption sensitive to the protein synthesis inhibitor cycloheximide was transiently increased by A23187, but returned to control levels within 30 min of incubation. These results suggest that elevation of intracellular Ca2+ leads to an energetic imbalance not related to stimulation of ATP consuming processes, but mainly due to impairment of mitochondrial function, possibly by the decoupling of oxidative phosphorylation and by inducing dissipative Ca2+ cycling.