In vivo and in vitro adenosine stimulation of ethanol oxidation by hepatocytes, and the role of the malate-aspartate shuttle

In this study, a pronounced increase of ethanol oxidation was found in hepatocytes obtained from adenosine-treated rats, or after in vitro additional of the nucleoside; this finding was accompanied by a maintenance of the normal cytoplasmic redox state. These results suggest a higher availability of cytoplasmic NAD in these cells. Therefore, the metabolic pathways which carry out the reoxidation of cytosolic reducing equivalents, namely, malate-aspartate and alpha-glycerophosphate shuttles, were examined. Isolated mitochondria from adenosine-treated rats had an increased NADH oxidation by the malate-aspartate shuttle; furthermore, in vivo and in vitro addition of adenosine to the hepatocytes induced changes in the equilibrium of the malate-aspartate shuttle, as evidenced by the subcellular distribution of the intermediates of this pathway. Acetaldehyde removal was also increased by adenosine and this fact was related to an elevated NAD/NADH ratio in the mitochondria. Thus, under these conditions, an increased ethanol uptake was accompanied by enhanced acetaldehyde removal in the animal. In conclusion, adenosine administration stimulates the transport of cytoplasmic reducing equivalents to the mitochondria, mainly through the malate-aspartate shuttle. This action, which may be located at the level of the mitochondrial membrane, is reflected by an enhancement of ethanol and acetaldehyde oxidations.     

In vivo evidence for the role of GM-CSF as a mediator in acute pancreatitis-associated lung injury

Severe pancreatitis is frequently associated with acute lung injury (ALI) and the respiratory distress syndrome. The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in mediating the ALI associated with secretagogue-induced experimental pancreatitis was evaluated with GM-CSF knockout mice (GM-CSF -/-). Pancreatitis was induced by hourly (12x) intraperitoneal injection of a supramaximally stimulating dose of the cholecystokinin analog caerulein. The resulting pancreatitis was similar in GM-CSF-sufficient (GM-CSF +/+) control animals and GM-CSF -/- mice. Lung injury, quantitated by measuring lung myeloperoxidase activity (an indicator of neutrophil sequestration), alveolar-capillary permeability, and alveolar membrane thickness was less severe in GM-CSF -/- than in GM-CSF +/+ mice. In GM-CSF +/+ mice, pancreas, lung and serum GM-CSF levels increase during pancreatitis. Lung levels of macrophage inflammatory protein (MIP)-2 are also increased during pancreatitis, but, in this case, the rise is less profound in GM-CSF -/- mice than in GM-CSF +/+ controls. Administration of anti-MIP-2 antibodies was found to reduce the severity of pancreatitis-associated ALI. Our findings indicate that GM-CSF plays a critical role in coupling pancreatitis to ALI and suggest that GM-CSF may act indirectly by regulating the release of other proinflammatory factors including MIP-2.     

In vivo and in vitro evidence concerning the role of lipid peroxidation in the mechanism of hepatocyte death due to carbon tetrachloride

Isolated rat hepatocytes exposed to CCl₄ showed a stimulated formation of malonaldehyde after only 30–60 min incubation. Conversely, the onset of hepatocyte death was a relatively late event, being significant only after 2–3 h of treatment. A cause–effect relationship between the two phenomena has been demonstrated by using hepatocytes isolated from rats pretreated with alpha-tocopherol. Comparable results were obtained in vivo where supplementation with alpha-tocopherol 15 h before CCl₄ dosing induced a partial or complete protection against the drug's necrogenic effect, depending on the concentration of the haloalkane used. Moreover, the vitamin supplementation prevented the CCl₄-induced increase of liver total calcium content, probably by blocking alterations in the liver cell plasma membranes due to lipid peroxidation.     

In vivo and in vitro evidence for ACh-stimulated L-arginine uptake

Whereas L-arginine is clearly recognized as the precursor for nitric oxide synthesis, and its entry into endothelial cells via system y(+) transport is established, few data exist regarding the acute regulation of this transport process. We specifically investigated the effect of ACh and isoprenaline (Iso) on L-arginine uptake in the human forearm and in cultured bovine aortic endothelial cells (BAEC). Sixteen healthy males were studied. During a steady-state intra-arterial infusion of [(3)H]L-arginine (100 nCi/min), the effects of ACh (9.25 and 37 microg/min), Iso (25-50 and 200 microg/min), and sodium nitroprusside (SNP) (1-2 and 8 microg/min) on forearm plasma flow (FPF), L-[(3)H]arginine uptake, and L-[(3)H]citrulline release were determined. In parallel experiments, the effects of ACh, Iso, and SNP on L-[(3)H]arginine uptake were studied in BAEC. L-Arginine uptake was inversely related to FPF (r = -0.50; P < 0.005). At a similar FPF (ACh 56.82 +/- 9.25, Iso 58.49 +/- 5.56, SNP 57.92 +/- 4.96 ml/min; P = ns), intra-arterial ACh significantly increased forearm uptake of L-[(3)H]arginine (54,655 +/- 8,018 dpm/min), compared with that observed with either Iso (40,517.23 +/- 6,841 dpm/min; P = 0.01) or SNP (36,816 +/- 4,650 dpm/min; P = 0.011). This was associated with increased ACh-induced L-[(3)H]citrulline release compared with Iso and SNP (P = 0.046). Similarly, in BAEC, ACh significantly increased L-[(3)H]arginine uptake compared with control, Iso, or SNP (ACh 12.0 x 10(7) +/- 1.83 x 10(7) vs. control 6.67 x 10(7) +/- 1.16 x 10(7) vs. Iso 7.35 x 10(7) +/- 1.63 x 10(7) vs. SNP 6.01 x 10(7) +/- 1.11 x 10(7) fmol.min(-1).mg(-1) at 300 micromol/l L-arginine; P = 0.043). Taken together, these data indicate that ACh stimulates L-arginine uptake in cultured endothelial cells and in human forearm circulation, indicating the potential for acute modulation of endothelial L-arginine uptake.     

In vivo evidence against a role for adenosine in the exercise pressor reflex in humans.

The pressor response to exercise is of great importance in both physiology and pathophysiology. Whether endogenous adenosine is a trigger for this reflex remains controversial. Muscle interstitial adenosine concentration can be determined by microdialysis. However, there are indications that local muscle cell damage by the microdialysis probe confounds these measurements in exercising muscle. Therefore, we used the nucleoside uptake inhibitor dipyridamole as pharmacological tool to bypass this confounding. We used microdialysis probes to measure endogenous adenosine in forearm skeletal muscle of healthy volunteers during two cycles of 15 min of intermittent isometric handgripping. During the second contraction, dipyridamole (12 microg.min(-1).dl forearm(-1)) was administered into the brachial artery. Dipyridamole potentiated the exercise-induced increase in dialysate adenosine from 0.30 +/- 0.08 to 0.48 +/- 0.10 micromol/l (n = 9, P < 0.05), but it did not potentiate the exercise-induced increase in blood pressure. A time-control study without dipyridamole revealed no difference in exercise-induced increase in adenosine between both contractions (n = 8). To exclude the possibility that the dipyridamole-induced increase in dialysate adenosine originates from extravasation of increased circulating adenosine, we simultaneously measured adenosine with microdialysis probes in forearm muscle and antecubital vein. In a separate group of nine volunteers, simultaneous intrabrachial infusion of 100 microg.min(-1).dl(-1) dipyridamole and 5 microg.min(-1).dl(-1) adenosine increased dialysate adenosine from the intravenous but not the interstitial probe, indicating preserved endothelial barrier function for adenosine. We conclude that dipyridamole significantly inhibits uptake of interstitial adenosine without affecting the pressor response to exercise, suggesting that interstitial adenosine is not involved in the pressor response to rhythmic isometric exercise.     

In vitro and in vivo inhibition of muscle lipid and protein oxidation by carnosine.

Carnosine, a beta-alanyl-L-histidine dipeptide with antioxidant properties is present at high concentrations in skeletal muscle tissue. In this study, we report on the antioxidant activity of carnosine on muscle lipid and protein stability from both in vitro and in vivo experiments. Carnosine inhibited lipid peroxidation and oxidative modification of protein in muscle tissue prepared from rat hind limb homogenates exposed to in vitro Fenton reactant (Fe2+, H2O2)-generated free radicals. The minimum effective concentrations of carnosine for lipid and protein oxidation were 2.5 and 1 mM, respectively. Histidine and beta-alanine, active components of carnosine, showed no individual effect towards inhibiting either lipid or protein oxidation. Skeletal muscle of rats fed a histidine supplemented diet for 13 days exhibited a marked increase in carnosine content with a concomitant reduction in muscle lipid peroxidation and protein carbonyl content in skeletal muscle caused by subjecting rats to a Fe-nitrilotriacetate administration treatment. This significant in vitro result confirms the in vivo antioxidant activity of carnosine for both lipid and protein constituents of muscle under physiological conditions.     

Transfer of cholesterol and diacylglycerol from lipophorin to Bombyx mori ovarioles in vitro: role of the lipid transfer particle

The objective of this study was to characterize the transfer of diacylglycerol (DAG) and cholesterol from larval Bombyx mori lipophorin to ovarioles. Transfer studies were carried out by incubating pupal ovarioles (5-day) with [(3)H]-cholesterol and [(3)H]-DAG-labeled lipophorin under different conditions. Transfer of both cholesterol and DAG exhibited hyperbolic dependency on lipophorin concentration with apparent Km values of 0.83 +/- 0.17 mg/ml and 0.74 +/- 0.16 mg/ml, respectively. Pretreatment of ovarioles with anti-lipid transfer particle (LTP) IgG significantly inhibited transfer of labeled DAG to ovarioles (75%) and not cholesterol. Injection of B. mori pupae (day 4) with anti-LTP IgG significantly affected the weight (65%), number of eggs (49%), amount of lipid (74%), and protein (65%) of the adult ovaries. Matured eggs had a very faint yellow color and deformed shape compared to controls. The inhibitory effect demonstrates the active role LTP plays in growth of ovaries, development, and oogenesis. The effect on vitellogenin shortage on egg development and maturation was determined by implanting ovaries in male recipients that lack vitellogenin. An 80% decline in egg production was observed. However, the mature eggs were normal in shape, color, and lipid content. Thus, restricting lipid or protein delivery to developing ovaries would dramatically affect choriogenesis.     

Anopheles gambiae lipophorin: characterization and role in lipid transport to developing oocyte

Lipid transport in arthropods is achieved by highly specialized lipoproteins, which resemble those described in vertebrate blood. Here, we describe purification and characterization of the lipid-apolipoprotein complex, lipophorin (Lp), in the malaria vector mosquito Anopheles gambiae. We also describe the Lp-mediated lipid transfer to developing eggs and the distribution of the imported lipid in developing embryos. The density of the Lp complex was 1.135 g/ml with an apparent molecular weight of 630 kDa. It is composed of two major polypeptides, apoLp I (260 kDa) and apoLp II (74 kDa) and composed of 50% protein, 48% lipid and 2% carbohydrate (w/w). Hydrocarbon, cholesterol, phosphatidyl choline, phosphatidyl ethanolamine, cholesteryl ester and diacylglyceride were the major Lp-associated lipids. Using fluorescently tagged lipids, we observed patterns that suggest that in live developing oocytes, the Lp was taken up by a receptor-mediated endocytic process. Such process was blocked at low temperature and in the presence of excess unlabeled Lp, but not by bovine serum albumin. Imported Lp was segregated in the spherical yolk bodies (mean size 1.8 microm) and distributed evenly in the cortex of the oocyte. In embryonic larvae, before hatching, a portion of the fatty acid in vesicles was found evenly distributed along the body, whereas portion of phospholipids was accumulated in the intestine.     

In vivo and in vitro protective role of proline

Accumulation of proline in response to environmental stresses seems tobe widespread among plants. To elucidate the role of proline in plantresponses,in vivo and in vitro, we studied theeffect of proline on catalase (CAT; EC 1.11.1.6), peroxidase (POD; EC 1.11.1.7)and polyphenol oxidase (PPO; EC 1.14.18.1). In vivo, thesethree enzymes were activated by proline, while CAT and POD were activated andPPO was inactivated by NaCl. In vitro, CAT and POD wereactivated and PPO was inactivated by proline. Proline appeared to protect thesethree enzyme activities. The significance of these findings with regard toenvironmental stress-induced proline accumulation in vivois discussed. The ability of proline to activate the enzymes may suggest alimited conformational change. These results are important for characterisationof metabolic responses to environmental stresses and can be used as a stressindicator.     

In vitro evidence indicating a role for the Fc region of IgG in the mechanism for the long-term maintenance and regulation of antibody levels in vivo

The synthesis of anti-human serum albumin (HSA) antibody was induced spontaneously in cell cultures prepared from the draining lymph nodes of rabbits immunized months earlier with polymerized HSA. Serum from HSA-immunized rabbits suppressed this response. Removal of specific antibody from immune serum eliminated suppression and the addition of specific IgG restored suppression, indicating that the feedback phenomenon may be explained by an effect of specific IgG antibody. Fab and F(ab′)₂ fragments masked antigen as effectively as IgG; however, they were markedly inferior to IgG in mediating suppression. Furthermore, Fab competed with IgG and interfered with IgG mediated suppression. The addition of small amounts of antigen to antibody-suppressed cultures induced an antibody response. The level of induction was proportional to the antigen-antibody ratio. However, 80 to 100 times the antibody concentration needed to mask all antigenic determinants was needed in order to eliminate induction of antibody synthesis. High concentrations of antigen-antibody complexes at equivalence also suppressed the spontaneous response. This suppression was similar to antibody mediated suppression at the spontaneous response in that the Fc region of IgG was required.     

Prostaglandin synthesis by fetal rat bone in vitro: evidence for a role of prostacyclin.

Prostaglandin synthesis by fetal rat bones was examined by thin-layer chromatography of culture media after preincubation with labeled arachidonic acid. Cultures in rabbit complement (non-heat inactivated serum) were compared with cultures in heat-inactivated serum or cultures treated with indomethacin. The major complement-dependent products were PGE2, PGF2 alpha and 6-keto-PGF1 alpha, the metabolite of prostacyclin (PGI2). Since PGI2 had not been previously identified in bone its ability to stimulate bone resorption was tested. Repeated addition of PGI2 stimulated release of previously incorporated 45Ca from fetal rat long bones in both short-term and long-term cultures at concentrations of 10(-5) to 10(-9)M. Because of the short half life of PGI2 in solution at neutral pH, we tested a sulfur analog, thiaprostacyclin (S-PGI2) which was found to be a stimulator of bone resorption at concentrations of 10(-5) to 10(-6)M. These studies suggest that endogenous PGI2 production may play a role in bone metabolism. Since vessels produce PGI2 it is possible that PGI2 release may be responsible for the frequent association between vascular invasion and resorption of bone or calcified cartilage in physiologic remodeling and pathologic osteolysis.     

In vitro and in vivo evidence that the C-terminus of preproenkephalin-A circulates as an 8500-dalton molecule

One of the major immunoreactive components of the enkephalin precursor BAM-22P in extracts of adrenal medulla is a molecule of approximately 8500 Da. A similar component is detected, after gel-permeation chromatography, in extracts of bovine plasma and in culture medium harvested from acetylcholine-stimulated bovine adrenomedullary cells in culture. Simultaneous detection of the C-terminal epitome of preproenkephalin-A: Met-enkephalin-Arg6-Phe7-OH in this secreted high-molecular-weight zone suggest that the approximately 8500-Da form of BAM-22P is the C-terminal of preproenkephalin A.     

The effect of adding a lipid tail to tyrosine-azobenzenearsonate: In vivo and in vitro consequences for cell-mediated immunity

Coupling lipid tails of increasing length to the tyrosine-azobenzene-arsonate molecule resulted in a decrease in in vivo immunogenicity. When used to induce in vitro stimulation of sensitized lymphocytes a decrease in dose was required to produce an optimal response as the lipid sidechain increased in size. This was attributable to the cytotoxic effects produced by the detergent-like activity of the large lipid side-chain derivatives. When used for pulsing of macrophages the stickiness of the larger lipid derivatives made them more effective at low doses and times for initiation of lymphocyte stimulation.     

Tyrosinase folding and copper loading in vivo: a crucial role for calnexin and alpha-glucosidase II.

Tyrosinase is the key enzyme of melanin biosynthesis. It is a multiply glycosylated metalloenzyme, which has a long maturation time making it an ideal in vivo model system to probe protein folding and metal loading events. The use of NB-DNJ, an alpha-glucosidase I and II inhibitor has allowed us to dissect these processes. Here we show that tyrosinase folds through several inactive intermediates, at least two of which are recognised by the ER chaperone, calnexin. If the association with calnexin is prevented, more rapid folding occurs, the resulting protein fails to bind copper and is inactive. If dissociation from calnexin is inhibited, folding is prevented; the protein does not go through the normal secretory pathway and is targeted for degradation. Thus, tyrosinase folds off calnexin, giving alpha-glucosidase II a critical role, but the association with calnexin is essential to promote the correct folding which enables it to acquire copper.     

In vivo and in vitro evidence of dopaminergic system down regulation induced by chronic L-DOPA

The biochemical modifications which occur in the dopaminergic system after chronic administration of L-DOPA are investigated. Levels of DA and of its metabolite 3-methoxytyramine (3-MT), an expression of the amount of DA released, were raised to the same extent in controls given a single dose of 1-DOPA and in chronically treated rats given 100 mg/kg of 1-DOPA plus 25 mg/kg of benserazide twice a day for 24 days. However, the reduction in neuronal function expressed by the decrease in 3-MT which follows treatment with DA agonists such as piribedil and apomorphine was less pronounced in the chronically L-DOPA treated rats. This suggests that such treatment causes a down regulation of DA receptors. These in vivo results were confirmed by in vitro analysis of DA receptor activity after chronic L-DOPA. Under these conditions there was a significant reduction in the number of [3H]-spiperone and [3H]-ADTN binding sites with no changes in their affinity. The in vivo and in vitro findings both suggest the involvement of a subsensitive compensatory mechanism or down regulation of dopaminergic neurons after chronic treatment with L-DOPA.     

In vitro evidence for senescent multinucleated melanocytes as a source for tumor-initiating cells

Oncogenic signaling in melanocytes results in oncogene-induced senescence (OIS), a stable cell-cycle arrest frequently characterized by a bi- or multinuclear phenotype that is considered as a barrier to cancer progression. However, the long-sustained conviction that senescence is a truly irreversible process has recently been challenged. Still, it is not known whether cells driven into OIS can progress to cancer and thereby pose a potential threat. Here, we show that prolonged expression of the melanoma oncogene N-RAS^61K in pigment cells overcomes OIS by triggering the emergence of tumor-initiating mononucleated stem-like cells from senescent cells. This progeny is dedifferentiated, highly proliferative, anoikis-resistant and induces fast growing, metastatic tumors. Our data describe that differentiated cells, which are driven into senescence by an oncogene, use this senescence state as trigger for tumor transformation, giving rise to highly aggressive tumor-initiating cells. These observations provide the first experimental in vitro evidence for the evasion of OIS on the cellular level and ensuing transformation.Cellular senescence is characterized by cell-cycle arrest and alterations in cell shape and metabolism, and can be triggered either by the sequential loss of telomeres or by numerous forms of cellular stress, for example, UV irradiation, oxidative stress or aberrant oncogenic signaling (premature senescence). In particular, oncogene-induced senescence (OIS), driven for example by activated RAS or BRAF, is an anti-cancer protection mechanism that prevents tumor generation despite the presence of oncogenic mutations. For instance, human nevi exhibit enhanced MAPK signaling caused by activating mutations in B-RAF or N-RAS. They display classical characteristics of senescence,¹ and remain benign in the large majority of cases. However, nevi are also supposed to give rise to a quarter of all melanomas.²Along the same lines, oncogenic RAS clearly triggers OIS in different cell types in vivo,^{3, 4, 5, 6} but activated RAS is detected in up to 30% of human cancers.^{7, 8} This indicates that senescence bypass is a key feature of cancer development. The fact that many premalignant tissues with tumorigenic potential display features of senescence has led to the concept that OIS precedes transformation, and tumors arise from senescent tissue.^{1, 5, 6, 9} However, as OIS was long considered to be irreversible, it was not clear how this transformation process can take place. Recently, there has been accumulating evidence that OIS can be reversed under certain circumstances on the cellular level. Specifically, H-RAS^12V induces senescence in fibroblasts, which is caused by ribonucleotide reductase (RRM2) suppression and accompanying dNTP reduction.¹⁰ Interestingly, the forced re-expression of RRM2 is able to overcome senescence. Still, is not known whether a senescent primary cell might give rise to cancer. While the later steps of tumor progression are fairly well understood, early events in tumorigenesis such as the transition of a benign senescent lesion to a tumor are still enigmatic.Here, we reveal that long-term NRAS^61K activation in melanocytes triggers a strong senescent phenotype characterized by multinucleation, which then is followed by the post-senescence generation of tumor-initiating cells with stem cell-like properties. The results demonstrate that senescence in melanocytes can be overcome on the cellular level and can also be a source for malignant cancer cells.     

In vivo and in vitro evidence for extracellular caspase activity released from apoptotic cells

While caspases play an established role as intracellular executors of apoptosis, little is known about extracellular activities of this ubiquitously expressed family of proteases. We demonstrate here that recombinant caspase-3 retained enzymatic activity in various extracellular fluids. Experiments with cell lines, primary cells, and mice with fulminant CD95-triggered hepatitis showed that significant amounts of DEVD-aminofluoromethylcoumarine-cleaving activity, indicative of active effector caspases, were released into the medium/plasma during apoptosis. Furthermore, caspase activities were detected in liquor samples from human head trauma patients. These findings warrant closer investigation of DEVDase activity as a diagnostic marker, and of potential extracellular substrates for caspases.