Effect of extracellular Ca2+ and Mg2+ on enzyme release from quiescent fibroblasts during various exposures

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.     

Release kinetics of ATP in cells exposed to nonionic detergents

We have previously shown that the protein binding of intracellular ATP could be examined by monitoring the ATP release kinetics from Triton X-100 and Brij 58 nonionic detergent permeabilized cells. We have now analysed the protein binding of ATP in an isotonic medium using intact and partially ATP depleted Brij 58 treated human erythrocytes. The effects of Triton X-100 below the critical micelle concentration (CMC) was studied in normal and tumorous tissue culture cells and human red blood cells. Our results showed that the protein association of ATP was altered in the partially ATP depleted erythrocytes. Below the CMC value, but above a critical level Triton X-100 treatment was effective in mobilizing the intracellular ATP in both cell types. The ATP release curves were sigmoidal and an ‘all or none’ type of response was observed, especially in erythrocytes. The use of Triton X-100 (< CMC) delays the detergent-induced cell decomposition time thus providing a new approach to investigating the physical state of intracellular ATP.     

Effects of lethal exposure to hyperoxia and to hydrogen peroxide on NAD(H) and ATP pools in Chinese hamster ovary cells

Cell death by oxidative stress has been proposed to be based on suicidal NAD depletion, typically followed by ATP depletion, caused by the NAD-consuming enzyme poly(ADP)ribose polymerase, which becomes activated by the presence of excessive DNA-strand breaks. In this study NAD+, NADH and ATP levels as well as DNA-strand breaks (assayed by alkaline elution) were determined in Chinese hamster ovary (CHO) cells treated with either H2O2 or hyperoxia to a level of more than 80% clonogenic cell killing. With H2O2 extensive DNA damage and NAD depletion were observed, while at a higher H2O2 dosage ATP also became depleted. In agreement with results of others, the poly(ADP)ribose polymerase inhibitor 3-aminobenzamide completely prevented NAD depletion. However, both H2O2-induced ATP depletion and cell killing were unaffected by the inhibitor, suggesting that ATP depletion may be a more critical factor than NAD depletion in H2O2-induced killing of CHO cells. With hyperoxia, only moderate DNA damage (2 X background) and no NAD depletion were observed, whereas ATP became largely (70%) depleted. We conclude that (1) there is no direct relation between ATP and NAD depletion in CHO cells subjected to toxic doses of H2O2 or hyperoxia; (2) H2O2-induced NAD depletion is not by itself sufficient to kill CHO cells; (3) killing of CHO cells by hyperoxia is not due to NAD depletion, but may be due to depletion of ATP.     

Importance of the cellular energy level for enzyme release induced by direct membrane damage

A release of intracellular enzymes may occur as a result of energy depletion of the cells or after direct membrane damage. A direct membrane damage, however, may be counteracted by the cell by energy-consuming reactions, thus more or less being dependent of the cellular energy level. Therefore, the association between enzyme release and the energy level was investigated after addition of various agents impairing the normal membrane function, i.e. lysophosphatidylcholine, phospholipases, the Ca ionophore A23187, ouabain and superoxide/H2O2, and after incubation in a hypotonic medium. It was observed that in some types of membrane damage the cellular energy is minimally involved, in other types the extent of enzyme release depends on the cellular energy level, and in some other types the cellular energy is affected but the connection to the enzyme release is not clear. The results also indicate that the effect of membrane-active agents arising in ATP-depleted states may be more severe in ATP-depleted than in normal cells.     

Gastric erosions following sympathetic denervation in the maternally deprived rat.

The adenosine triphosphate (ATP) content of rat mast cells was studied during and after histamine release induced by compound 48/80. The almost identical time course of ATP decrease from mast cells treated with either glycolytic or respiratory inhibitors seems to indicate that the ATP depletion was largely related to the histamine release process and not to an uncoupling of the oxidative phosphorylation. These results support the view that histamine release induced by compound 48/80 is an energy-requiring process. The ATP content of the cells was not, however, restored within the two hours of observation. The cause of the prolonged decrease in the ATP level has been discussed.     

Effect of oxygen tension on human peripheral blood leukocytes: lysosomal enzyme release and metabolic responses during phagocytosis

We found that nonlethal lysosomal enzyme release from human peripheral blood leukocytes during phagocytosis of opsonized zymosan in vitro was modified by the oxygen tension under which the cells were incubated; with decreasing Po(2), zymosan-induced release of lysosomal enzymes was potentiated. The effect on enzyme release could not be attributed secondarily to an effect on phagocytosis, because, as others have reported, Po(2) had little effect on that response. Metabolic responses that accompany phagocytosis were also modified by oxygen tension. Stimulation of oxidation by way of the pentose cycle was further enhanced by increasing Po(2). Conversely, anaerobic glycolysis was promoted by decreasing oxygen tension. ATP levels fell as a function of time and concentration of phagocytic stimulus, mirroring lysosomal enzyme release as modified by Po(2). Cyclic AMP levels fell during phagocytosis and lysosomal enzyme release, a change that could act to facilitate lysosomal enzyme release. However, the fall in nucleotide level was greatest with highest Po(2) (i.e., when lysosomal enzyme release was least). The inverse relationship between oxidative metabolism and enzyme release suggested that a product of oxidative metabolism might adversely influence enzyme release. Sulfhydryl antioxidants (Cysteine, glutathione) and scavengers of oxygen-derived reactants (superoxide dismutase, catalase, benzoate, hypoxanthine, xanthine, histidine, azide) all potentiated zymosan- stimulated enzyme release. These findings are consistent with the interpretation that one or more factors (e.g., superoxide anion, hydrogen peroxide, hydroxyl radical, singlet oxygen), generated in association with the burst of oxidative metabolism which accompanies phagocytosis, acts to inhibit lysosomal enzyme release.     

Bcl-2 prevents Bax oligomerization in the mitochondrial outer membrane

ATP depletion results in Bax translocation from cytosol to mitochondria and release of cytochrome c from mitochondria into cytosol in cultured kidney cells. Overexpression of Bcl-2 prevents cytochrome c release, without ameliorating ATP depletion or Bax translocation, with little or no association between Bcl-2 and Bax as demonstrated by immunoprecipitation (Saikumar, P., Dong, Z., Patel, Y., Hall, K., Hopfer, U., Weinberg, J. M., and Venkatachalam, M. A. (1998) Oncogene 17, 3401-3415). Now we show that translocated Bax forms homo-oligomeric structures, stabilized as chemical adducts by bifunctional cross-linkers in ATP-depleted wild type cells, but remains monomeric in Bcl-2-overexpressing cells. The protective effects of Bcl-2 did not require Bcl-2/Bax association, at least to a degree of proximity or affinity that was stable to conditions of immunoprecipitation or adduct formation by eight cross-linkers of diverse spacer lengths and chemical reactivities. On the other hand, nonionic detergents readily induced homodimers and heterodimers of Bax and Bcl-2. Moreover, associations between translocated Bax and the voltage-dependent anion channel protein or the adenine nucleotide translocator protein could not be demonstrated by immunoprecipitation of Bax, or by using bifunctional cross-linkers. Our data suggest that the in vivo actions of Bax are at least in part dependent on the formation of homo-oligomers without requiring associations with other molecules and that Bcl-2 cytoprotection involves mechanisms that prevent Bax oligomerization.     

Guanine nucleotides modulate the effects of brefeldin A in semipermeable cells: regulation of the association of a 110-kD peripheral membrane protein with the Golgi apparatus

The release of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A (BFA) action, preceding the movement of Golgi membrane into the ER. ATP depletion also causes the reversible redistribution of the 110-kD protein from Golgi membrane into the cytosol, although no Golgi disassembly occurs. To further define the effects of BFA on the association of the 110-kD protein with the Golgi apparatus we have used filter perforation techniques to produce semipermeable cells. All previously observed effects of BFA, including the rapid redistribution of the 110-kD protein and the movement of Golgi membrane into the ER, could be reproduced in the semipermeable cells. The role of guanine nucleotides in this process was investigated using the nonhydrolyzable analogue of GTP, GTP gamma S. Pretreatment of semipermeable cells with GTP gamma S prevented the BFA-induced redistribution of the 110-kD protein from the Golgi apparatus and movement of Golgi membrane into the ER. GTP gamma S could also abrogate the observed release of the 110-kD protein from Golgi membranes which occurred in response to ATP depletion. Additionally, when the 110-kD protein had first been dissociated from Golgi membranes by ATP depletion, GTP gamma S could restore Golgi membrane association of the 110-kD protein, but not if BFA was present. All of these effects observed with GTP gamma S in semipermeable cells could be reproduced in intact cells treated with AlF4-. These results suggest that guanine nucleotides regulate the dynamic association/dissociation of the 110-kD protein with the Golgi apparatus and that BFA perturbs this process by interfering with the association of the 110-kD protein with the Golgi apparatus.     

ATP: a mediator for HCl-induced TRPV1 activation in esophageal mucosa

In esophageal mucosa, HCl causes TRPV1-mediated release of calcitonin gene-related peptide (CGRP) and substance P (SP) from submucosal neurons and of platelet-activating factor (PAF) from epithelial cells. CGRP and SP release was unaffected by PAF antagonists but reduced by the purinergic antagonist suramin. ATP caused CGRP and SP release from esophageal mucosa, confirming a role of ATP in the release. The human esophageal epithelial cell line HET-1A was used to identify epithelial cells as the site of ATP release. HCl caused ATP release from HET-1A, which was reduced by the TRPV1 antagonist 5-iodoresiniferatoxin. Real-time PCR demonstrated the presence of mRNA for several P2X and P2Y purinergic receptors in epithelial cells. HCl also increased activity of lyso-PAF acetyl-CoA transferase (lyso-PAF AT), the enzyme responsible for production of PAF. The increase was blocked by suramin. ATP caused a similar increase, confirming ATP as a mediator for the TRPV1-induced increase in enzyme activity. Repeated exposure of HET-1A cells to HCl over 2 days caused upregulation of mRNA and protein expression for lyso-PAF AT. Suramin blocked this response. Repeated exposure to ATP caused a similar mRNA increase, confirming ATP as a mediator for upregulation of the enzyme. Thus, HCl-induced activation of TRPV1 causes ATP release from esophageal epithelial cells that causes release of CGRP and SP from esophageal submucosal neurons and activation of lyso-PAF AT, the enzyme responsible for the production of PAF in epithelial cells. Repeated application of HCl or of ATP causes upregulation of lyso-PAF AT in epithelial cells.     

Human immunodeficiency virus type 1 pathogenesis in SCID-hu mice correlates with syncytium-inducing phenotype and viral replication

Human immunodeficiency virus type 1 (HIV-1) patient isolates and molecular clones were used to analyze the determinants responsible for human CD4(+) thymocyte depletion in SCID-hu mice. Non-syncytium-inducing, R5 or R3R5 HIV-1 isolates from asymptomatic infected people showed little or no human CD4(+) thymocyte depletion in SCID-hu mice, while syncytium-inducing (SI), R5X4 or R3R5X4 HIV-1 isolates from the same individuals, isolated just prior to the onset of AIDS, rapidly and efficiently eliminated CD4-bearing human thymocytes. We have mapped the ability of one SI HIV-1 isolate to eliminate CD4(+) human cells in SCID-hu mice to a region of the env gene including the three most amino-terminal variable regions (V1 to V3). We find that for all of the HIV-1 isolates that we studied, a nonlinear relationship exists between viral replication and the depletion of CD4(+) cells. This relationship can best be described mathematically with a Hill-type plot indicating that a threshold level of viral replication, at which cytopathic effects begin to be seen, exists for HIV-1 infection of thymus/liver grafts in SCID-hu mice. This threshold level is 1 copy of viral DNA for every 11 cells (95% confidence interval = 1 copy of HIV-1 per 67 cells to 1 copy per 4 cells). Furthermore, while SI viruses more frequently achieve this level of replication, replication above this threshold level correlates best with cytopathic effects in this model system. We used GHOST cells to map the coreceptor specificity and relative entry efficiency of these early- and late-stage patient isolates of HIV-1. Our studies show that coreceptor specificity and entry efficiency are critical determinants of HIV-1 pathogenesis in vivo.     

Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. ATP depletion blocks receptor recycling but not a single round of endocytosis

Continuous endocytosis of 125I-asialo-orosomucoid (ASOR) mediated by the galactosyl receptor in rat hepatocytes is a cyclic process. 125I-ASOR-receptor complexes are internalized, processed, and the ligand is degraded while the receptor is returned to the cell surface for reutilization. Since a true cycle has a thermodynamic requirement for the input of external energy, we examined the effects of changes in intracellular ATP levels on the function of the receptor cycle. Hepatocytes were depleted of ATP to various extents prior to endocytosis by incubating cells at 15 degrees C in the presence of 2 mM NaF and 0-20 mM NaN3. A luciferase-luciferin bioluminescence assay was used to quantitate the amount of cellular ATP. ATP-depleted cells were allowed to bind 125I-ASOR at 0 degrees C, washed through discontinuous Percoll gradients, and only viable cells were isolated and incubated at 37 degrees C to initiate a synchronous single round of endocytosis. The extent of internalization of this surface-bound 125I-ASOR was unaffected by an ATP depletion to less than 1% of the control level. The rate of internalization of surface-bound ligand was unaffected until the ATP levels decreased to 30% or less; at greater than 98% ATP depletion the initial rate decreased by a maximum of 55% and the kinetics became biphasic. In contrast, continuous endocytosis in the presence of excess ASOR was inhibited by only a 25% decline in cellular ATP content and demonstrated a very sharp threshold response to changing ATP levels. Continuous endocytosis, which requires receptor recycling, was completely inhibited when the total cellular ATP level decreased by only 40%. We conclude that the internalization phase of endocytosis is not dependent on ATP but that the processing and/or externalization phases of the complete receptor cycle are either directly or indirectly dependent on ATP and very sensitive to changes in cellular ATP content.     

Mitochondria-mediated ATP depletion by anti-cancer agents of the jasmonate family

Jasmonates are plant stress hormones that induce suppression of proliferation and death in cancer cells, while being selectively inactive towards non-transformed cells. Jasmonates can overcome apoptotic blocks and exert cytotoxic effects on drug-resistant cells expressing p53 mutations. Jasmonates induce a rapid depletion of ATP in cancer cells. Indeed, this steep drop occurs when no signs of cell death are detectable yet. Experiments using modulators of ATP synthesis via glycolysis or oxidative phosphorylation suggest that the latter is the pathway suppressed by jasmonates. Consequently, the direct effects of jasmonates on mitochondria were evaluated. Jasmonates induced cytochrome c release and swelling in mitochondria isolated from cancer cells but not from normal ones. Thus, the selectivity of jasmonates against cancer cells is rooted at the mitochondrial level, and probably exploits differences between mitochondria from normal versus cancer cells. These findings position jasmonates as promising anti-cancer drugs acting via energetic depletion in neoplastic cells.     

Adrenal glucocorticoids, adenine nucleotide translocation, and mitochondrial calcium accumulation.

The administration of dexamethasone to rats markedly diminished the initial rate and maximal extent of substrate-dependent calcium uptake in subsequently isolated liver mitochondria, and enhanced the release of calcium. The apparent Km for calcium transport was not altered by dexamethasone treatment and it ranged from 50 to 80 muM when an EDTA/Ca buffer system was used in the presence of magnesium, and 20 muM when an NTA/Ca buffer system without magnesium was employed. In contrast, when ATP was employed as the energy source, there was no significant difference in initial rate, Km, or the extent of calcium accumulation between mitochondria from control and dexamethasone-treated animals. Although mitochondria from dexamethasone-treated animal showed 15% less cytochrome c oxidase activity/mg of protein, overall respiratory capacity and ATP production from ADP were the same as in control mitochondria. However, mitochondria from dexamethasone-treated animals translocated ATP from inside to outside faster than those from control animals. When the ATP in the medium was depleted by glucose and hexokinase, both types of mitochondria retained essentially all the preloaded calcium until total ATP reached a critical level (7 approximately 5 mumol of ATP/mg of protein). When ATP content fell below this critical level, mitochondria released all the calcium quickly. Dexamethasone treatment increased the susceptibility of mitochondria to the depletion of ATP. These data indicate that the dexamethasone-induced decrease in maximal calcium transport and in calcium retention carrier system per se, but o an altered ability of the mitochondria to regulate intramitochondrial ATP content.     

Role of mitochondria-associated hexokinase II in cancer cell death induced by 3-bromopyruvate

It has long been observed that cancer cells rely more on glycolysis to generate ATP and actively use certain glycolytic metabolic intermediates for biosynthesis. Hexokinase II (HKII) is a key glycolytic enzyme that plays a role in the regulation of the mitochondria-initiated apoptotic cell death. As a potent inhibitor of hexokinase, 3-bromopyruvate (3-BrPA) is known to inhibit cancer cell energy metabolism and trigger cell death, supposedly through depletion of cellular ATP. The current study showed that 3-BrPA caused a covalent modification of HKII protein and directly triggered its dissociation from mitochondria, leading to a specific release of apoptosis-inducing factor (AIF) from the mitochondria to cytosol and eventual cell death. Co-immunoprecipitation revealed a physical interaction between HKII and AIF. Using a competitive peptide of HKII, we showed that the dissociation of hexokinase II from mitochondria alone could cause apoptotic cell death, especially in the mitochondria-deficient ρ⁰ cells that highly express HKII. Interestingly, the dissociation of HKII itself did not directly affect the mitochondrial membrane potential, ROS generation, and oxidative phosphorylation. Our study suggests that the physical association between HKII and AIF is important for the normal localization of AIF in the mitochondria, and disruption of this protein complex by 3-BrPA leads to their release from the mitochondria and eventual cell death.     

Interaction of protein kinase C with membranes is regulated by Ca2+, phorbol esters, and ATP

Physiologic regulation of protein kinase C activity requires its interaction with cellular membranes. We have recently shown that binding of the enzyme to plasma membranes is controlled by Ca2+, whereas enzyme activators, like phorbol esters, regulate both membrane binding and enzyme activity. Here we describe the factors which control the dissociation of protein kinase C from the plasma membrane. In the absence of phorbol esters, the dissociation reaction is rapid and is determined by varying the Ca2+ concentration between 0.1 and 1 microM. However, the presence of 4-beta-phorbol 12,13-dibutyrate greatly reduces enzyme release in response to Ca2+ depletion; removal of the phorbol ester itself permits efficient membrane-enzyme dissociation. The stabilization of the membrane-protein kinase C complex by phorbol esters can be reversed by ATP with an apparent Km for the nucleotide of 6.5 microM. The ATP effect requires MgCl2 and cannot be reproduced by other nucleotides or by a nonhydrolyzable analogue, suggesting that an ATP-dependent phosphorylation reaction may be involved. 4-beta-Phorbol 12,13-dibutyrate appears to stabilize membrane-enzyme association by reducing the apparent Km for Ca2+ to about 15 nM, whereas ATP reverses the phorbol ester effect by increasing the Km for Ca2+ to about 760 nM. Furthermore, the strong degree of negative cooperativity displayed by the Ca2+-dependent enzyme-membrane dissociation is consistent with the presence of multiple interacting Ca2+-binding sites on protein kinase C.     

Complex dynamics of chaperone-protein interactions under cellular stress

We present a model of a generalizable but minimalistic network based on the properties of interactions between proteins, molecular chaperones (e.g., Hsp 70, BiP) and ATP inside cells and subcellular components such as endoplasmic reticulum (ER). The dynamics of chaperone-dependent protein folding and misfolding in the cell can be modeled mathematically as a “predator-prey” problem, which can then be used to analyze the behavior of the system under conditions simulating stress (e.g., cardiac ischemia). We have tested this model under normal physiological and diseased conditions (e.g., ischemia as simulated by ATP depletion) and analyzed the effects of induction of chaperones (e.g., heat shock, tunicamycin) and inhibition of the degradative pathway (e.g., proteasome inhibition) on this model. Simulation gave the following results: (1) Under normal physiological conditions (basal levels of ATP, chaperone, and initially misfolded proteins), as expected, the model predicts the stable production of correctly folded proteins. (2) A threshold of ATP levels exists below which the system tends toward increasing degrees of complex behavior. When ATP levels are just above this threshold, the system is highly vulnerable to sudden, brief drops in ATP levels such as may occur in the setting of acute ischemia: bursts of oscillations continue even when ATP levels revert to the threshold. However, if ATP levels are rapidly increased to levels considerably above the threshold, the system becomes stable again. (3) Up to 10-fold increases in chaperone levels, such as those that occur under conditions of prior heat shock or (in the case of ER chaperones) tunicamycin treatment, did not affect the behavior of the system under basal conditions, nor did it affect the tendency to complex behavior in the setting of ATP depletion. It did, however, shorten the recovery period of the system after chaotic-type oscillations were induced by acute ATP depletion. (4) Blocking the degradative pathway for misfolded proteins (e.g., proteasome inhibition) predisposes the system toward instability in the setting of ATP depletion by changing the ATP threshold at which bursts of oscillations occur. These results support the hypothesis that there are distinct thresholds for ATP, chaperones, and degradative activity, outside which cellular protein folding dynamics become unstable. They also suggest that an important mechanism by which chaperone induction protects cells from subsequent stress is by limiting the tendency to instability after an insult (e.g., acute myocardial ischemia or acute tubular injury to the kidney). Thus, the model may be useful for understanding cell and tissue tolerance to stress and injury.     

Mitochondrial damage and its role in causing hepatocyte injury during stimulation of lipid peroxidation by iron nitriloacetate.

Incubation of isolated rat hepatocytes with 0.1 mM iron nitrilotriacetic acid (FeNTA) caused a rapid rise in lipid peroxidation followed by a substantial increase in trypan blue staining and lactate dehydrogenase release, but did not affect the protein and non-protein thiol content of the cells. Hepatocyte death was preceded by the decline of mitochondrial membrane potential, as assayed by rhodamine 123 uptake, and by the depletion of cellular ATP. Chelation of extracellular Ca2+ by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid or inhibition of Ca2+ cycling within the mitochondria by LaCl3 or cyclosporin A did not prevent the decline of rhodamine 123 uptake. On the other hand, a dramatic increase in the conjugated diene content was observed in mitochondria isolated from FeNTA-treated hepatocytes. Oxidative damage of mitochondria was accompanied by the leakage of matrix enzymes glutamic oxalacetic aminotransferase (GOT) and glutamate dehydrogenase (GLDH). The addition of the antioxidant N,N'-diphenylphenylene diamine (DPPD) completely prevented GOT and GLDH leakage, inhibition of rhodamine 123 uptake, and ATP depletion induced by FeNTA, indicating that Ca(2+)-independent alterations of mitochondrial membrane permeability consequent to lipid peroxidation were responsible for the loss of mitochondrial membrane potential. DPPD addition also protected against hepatocyte death. Similarly hepatocytes prepared from fed rats were found to be more resistant than those obtained from starved rats toward ATP depletion and cell death caused by FeNTA, in spite of undergoing a comparable mitochondrial injury. A similar protection was also observed following fructose supplementation of hepatocytes isolated from starved rats, indicating that the decline of ATP was critical for the development of FeNTA toxicity. From these results it was concluded that FeNTA-induced peroxidation of mitochondrial membranes impaired the electrochemical potential of these organelles and led to ATP depletion which was critical for the development of irreversible cell injury.     

Externalization of membrane-bound activities during sheep reticulocyte maturation is temperature and ATP dependent

During the maturation of sheep reticulocytes in vitro, there is release of material that can be pelleted from the cell-free incubation medium by centrifugation at 100,000 X g. This pellet contains activities that are derived from both the plasma membrane and lysosomes. No evidence was obtained for the presence of mitochondrial activities or cytosolic enzyme activities. The release of these activities is ATP and temperature dependent, since reduction of either results in a greater retention of the activities by the cells and a lesser amount in the 100,000 X g pellet. The pelleted material is vesicular in nature, and the production and (or) release of the material are reduced upon ATP depletion or lowering of the temperature. It is concluded that the externalization of specific membrane components is a normal metabolic process that occurs during reticulocyte maturation and represents a means by which reticulocytes shed specific types of membrane-associated functions that are known to decrease during reticulocytes maturation.     

Biosynthesis of paf-acether. IX. Role for a phosphorylation-dependent activation of acetyltransferase in antigen-stimulated mouse mast cells

Mouse bone marrow-derived mast cells passively sensitized with monoclonal IgE released paf-acether (platelet-activating factor) and beta-hexosaminidase when challenged with the specific antigen. The formation and the release of paf-acether followed an early increase in the activity of the acetyltransferase, the main enzyme in paf-acether biosynthesis. The antigen-induced activation of the acetyltransferase was dependent on physiologic temperature and on the presence of Ca2+. By using microsomal fractions from unchallenged and challenged mast cells, the Vmax values were 3.5 and 12.0 nmol/min/mg of protein, respectively, whereas in both cases a Km value for acetyl-coenzyme A of 172 microM was measured. The stimulation of acetyltransferase could be mimicked in vitro under experimental conditions which favor phosphorylation, i.e. adding ATP and Mg2+ to lysates from unchallenged mast cells. In contrast, ATP and Mg2+ were uneffective on lysates from challenged cells that exhibited high level of acetyltransferase activity, suggesting that phosphorylation of the enzyme already took place at the time of cell stimulation. Moreover, addition of alkaline phosphatase to microsomal fraction obtained from either antigen-challenged mouse bone marrow-derived mast cells or unchallenged cells, resulted in 52% and 43% loss of acetyltransferase activity, respectively. Phorbol myristate acetate treatment of cells doubled the enzyme activity supporting the phosphorylation hypothesis. Thus, we report on the immunologic activation of a key enzyme for paf-acether synthesis and on the mechanism of this activation in a pure mast cell population. A link between bridging of IgE receptors and the activation of an enzyme critical to the formation of a lipid mediator is thereby evidenced.     

Release of spectrin-free vesicles from human erythrocytes during ATP depletion: 1. characterization of spectrin-free vesicles

Human erythrocytes incubated without glucose at 37 degrees C (in vitro aging) release spectrin-free vesicles after 12 or more hours. The release of vesicles is dependent upon ATP depletion. If the endogenous level of ATP is maintained, vesicle release is completely inhibited up to 54 h. Vesicle release is independent of hemolysis because in vitro aged cells and cells that maintain their ATP levels lose identical amounts of hemoglobin up to 45 h. 93 percent of all membrane particles released constitute a uniform population of spheres with a diameter of 185 +/- 23nm. These vesicles are of slightly varying densities due to varying contents of hemoglobin. Vesicles contain half the amount of membrane protein that is found in intact membranes when referred to the content of phospholipids phosphorus. This is primarily due to the absence of spectrin. However, their content of protein component III, glycophorin, and cholesterol remains the same as in intact membranes. Thus, the major integral membrane proteins are present in vesicles in similar quantities were surface area as in cells except for the enzyme acetylcholinesterase that is enriched up to twofold. The phospholipids composition of these vesicles is representative of the intact membrane except that the amount of phosphatidic acid is 10-fold higher and the amount of phosphatidylethanolamine is slightly lower than in erythrocytes. These results suggest a selective release of membrane domains that lack peripheral membrane proteins and are enriched in acetylcholinesterase. This release of spectrin-free vesicles from cells aged in vitro could represent an acceleration of the physiological aging process.