Rate-limiting factors in the oxidation of ethanol by isolated rat liver cells.
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The oxidation of ethanol by isolated liver cells from starved rats is limited by the rate of removal of reducing equivalents generated in the cytosol by alcohol dehydrogenase. Evidence is presented suggesting that, in these cells, transfer of reducing equivalents from the cytosol to the mitochondria is regulated by the intracellular concentrations of the intermediates of the malate-aspartate and glycerol 3-phosphate cycles, as well as by flux through the respiratory chain. In liver cells isolated from fed rats, the availability of substrate increased the cell content of intermediates of the hydrogen-transfer cycles, and enhanced ethanol uptake. Under these conditions, ethanol consumption is limited by the availability of ADP for oxidative phosphorylation. (+info)
Role of anion translocation across the mitochondrial membrane in the regulation of urea synthesis from ammonia by isolated rat hepatocytes.
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The regulation of urea synthesis from ammonia was investigated using isolated hepatocytes from fasted rats. Addition of ammonia alone produced only a small increase of urea formation, which was stimulated 2-fold by ornithine in conjunction with a fall of ATP levels and an accumulation of citrulline. Further addition of oleate or beta-hydroxybutyrate produced an additional 2-fold stimulation of urea formation to approximately 200 mumol/g dry weight/hour. The presence of oleate also protected against the inhibitory effect of 2,4-dinitrophenol on urea synthesis and the cellular ATP content. The data suggest that both the rate of of energy production and the rate of generation of reducing equivalents from endogensou substrates are insufficient to meet the requirements for optimal rates of urea synthesis. Urea formation from NH3 in the presence of ornithine and oleate, but iin the absence of gluconeogenic precursors, was inhibited by butylmalonate, a known inhibitor of malate-phosphate exchange across the mitochondrial membrane, and stimulated by theaddition of malate and other dicarboxylic acids and amino acids to the cell suspension... (+info)
Facilitation of conditioned fear extinction by systemic administration or intra-amygdala infusions of D-cycloserine as assessed with fear-potentiated startle in rats.
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NMDA receptor antagonists block conditioned fear extinction when injected systemically and also when infused directly into the amygdala. Here we evaluate the ability of D-cycloserine (DCS), a partial agonist at the strychnine-insensitive glycine-recognition site on the NMDA receptor complex, to facilitate conditioned fear extinction after systemic administration or intra-amygdala infusions. Rats received 10 pairings of a 3.7 sec light and a 0.4 mA footshock (fear conditioning). Fear-potentiated startle (increased startle in the presence vs the absence of the light) was subsequently measured before and after 30, 60, or 90 presentations of the light without shock (extinction training). Thirty non-reinforced light presentations produced modest extinction, and 60 or 90 presentations produced nearly complete extinction (experiment 1). DCS injections (3.25, 15, or 30 mg/kg) before 30 non-reinforced light exposures dose-dependently enhanced extinction (experiment 2) but did not influence fear-potentiated startle in rats that did not receive extinction training (experiment 3). These effects were blocked by HA-966, an antagonist at the glycine-recognition site (experiment 4). Neither DCS nor HA-966 altered fear-potentiated startle when injected before testing (experiment 5). The effect of systemic administration was mimicked by intra-amygdala DCS (10 microg/side) infusions (experiment 6). These results indicate that treatments that promote NMDA receptor activity after either systemic or intra-amygdala administration promote the extinction of conditioned fear. (+info)
De novo-synthesized ceramide is involved in cannabinoid-induced apoptosis.
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Delta(9)-Tetrahydrocannabinol (THC) and other cannabinoids have been shown to induce apoptosis of glioma cells via ceramide generation. In the present study, we investigated the metabolic origin of the ceramide responsible for this cannabinoid-induced apoptosis by using two subclones of C6 glioma cells: C6.9, which is sensitive to THC-induced apoptosis; and C6.4, which is resistant to THC-induced apoptosis. Pharmacological inhibition of ceramide synthesis de novo, but not of neutral and acid sphingomyelinases, prevented THC-induced apoptosis in C6.9 cells. The activity of serine palmitoyltransferase (SPT), which catalyses the rate-limiting step of ceramide synthesis de novo, was remarkably enhanced by THC in C6.9 cells, but not in C6.4 cells. However, no major changes in SPT mRNA and protein levels were evident. Changes in SPT activity paralleled changes in ceramide levels. Pharmacological inhibition of ceramide synthesis de novo also prevented the stimulation of extracellular-signal-regulated kinase and the inhibition of protein kinase B triggered by cannabinoids. These findings show that de novo-synthesized ceramide is involved in cannabinoid-induced apoptosis of glioma cells. (+info)
Preparation of protoplasts and whole cell ghosts from Mycobacterium smegmatis.
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Cell wall-deficient forms of Mycobacterium smegmatis were produced in growth medium containing D-cycloserine and horse serum. These cells were transformed into protoplasts with EDTA and lysozyme. Subsequent lysis by nucleases followed by osmotic shock produced membrane vesicles (whole cell ghosts). (+info)
Cycloserine fatty acid derivatives as prodrugs: synthesis, degradation and in vitro skin permeability.
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Various 4,5-dihydroisoxazol-3-yl fatty acid ester derivatives of cycloserine were synthesized to improve skin permeation of cycloserine. The ester derivatives were prepared by using the tert-butoxycarbonyl (t-Boc) protection strategy. The 4,5-dihydroisoxazol-3-yl esters were readily hydrolysed in an aqueous buffer solution, and the degradation profiles showed both specific acid and specific base catalysis. In 50% human serum the formation of cycloserine was observed, but enzymatic catalysis was limited. Delivery through hairless mouse skin was investigated, and the apparent permeability coefficient was measured based on the flux of cycloserine into the receptor phase. The skin permeation of cycloserine across the hairless mouse skin was increased up to 20-fold by the fatty acid esters. The 4,5-dihydroisoxazol-3-yl fatty acid esters of cycloserine can therefore be considered as new topical prodrugs with the potential use in treatment of various skin infections. (+info)
Direct calcium binding results in activation of brain serine racemase.
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Serine racemase (SR) is a brain enzyme present in glial cells, where it isomerizes L-serine into D-serine that, in turn, diffuses and coactivates the N-methyl-D-aspartate receptor through the binding to the so-called "glycine site." We have developed a method for the slow expression of SR in a eukaryotic vector that permits the correct insertion of the prosthetic group into the active site, rendering functional SR with a K(m) toward L-serine of 4.8 mm. Divalent cations such as calcium or manganese were necessary for complete enzyme activity, whereas the presence of chelators such as EDTA completely inhibited the enzyme. Moreover, direct binding of calcium to SR was evidenced using (45)Ca(2+). Gel filtration of the recombinant SR revealed the protein to be in a dimer-tetramer equilibrium. The addition of EDTA to a calcium-saturated serine racemase evokes a profound conformational change, as monitored by both fluorescence and circular dichroism techniques. Fluorescence titration allowed us to calculate a binding constant for calcium of 6.2 microm. Reagents that react with sulfhydryl groups, such as cystamine, were potent inhibitors of SR, in a clear reflection that one or more cysteine residues are important for enzyme activity. Additionally, 16 serine analogues were tested as a putative SR substrate or inhibitors. Significant inhibition was only observed for L-Ser-O-sulfate, L-cycloserine, and L-cysteine. Finally, activation of brain SR as a result of the changes in calcium concentration was studied in primary astrocytes. Treatment of astrocytes with the calcium ionophore, as well as with compounds that augment the intracellular calcium levels such as glutamate or kainate led to an increase in the amount of d-serine present in the extracellular medium. These results suggest that there might be a glutamatergic-mediated regulation of SR activity by intracellular calcium concentration. (+info)
Ceramide signaling in fenretinide-induced endothelial cell apoptosis.
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Stress stimuli can mediate apoptosis by generation of the lipid second messenger, ceramide. Herein we investigate the molecular mechanism of ceramide signaling in endothelial apoptosis induced by fenretinide (N-(4-hydroxyphenyl)retinamide (4-HPR)). 4-HPR, a synthetic derivative of retinoic acid that induces ceramide in tumor cell lines, has been shown to have antiangiogenic effects, but the molecular mechanism of these is largely unknown. We report that 4-HPR was cytotoxic to endothelial cells (50% cytotoxicity at 2.4 microm, 90% at 5.36 microm) and induced a caspase-dependent endothelial apoptosis. 4-HPR (5 microm) increased ceramide levels in endothelial cells 5.3-fold, and the increase in ceramide was required to achieve the apoptotic effect of 4-HPR. The 4-HPR-induced increase in ceramide was suppressed by inhibitors of ceramide synthesis, fumonisin B(1), myriocin, and l-cycloserine, and 4-HPR transiently activated serine palmitoyltransferase, demonstrating that 4-HPR induced de novo ceramide synthesis. Sphingomyelin levels were not altered by 4-HPR, and desipramine had no effect on ceramide level, suggesting that sphingomyelinase did not contribute to the 4-HPR-induced ceramide increase. Finally, the pancaspase inhibitor, t-butyloxycarbonyl-aspartyl[O-methyl]-fluoromethyl ketone, suppressed 4-HPR-mediated apoptosis but not ceramide accumulation, suggesting that ceramide is upstream of caspases. Our results provide the first evidence that increased ceramide biosynthesis is required for 4-HPR-induced endothelial apoptosis and present a molecular mechanism for its antiangiogenic effects. (+info)