Metabolism and salvage of adenine and hypoxanthine by myocytes isolated from mature rat heart

Adenine and hypoxanthine can be utilised by cardiac muscle cells as substrates for the synthesis of ATP. A possible therapeutic advantage of these compounds as high-energy precursors is their lack of vasoactive properties. Myocytes isolated from mature rat heart have been used to establish in kinetic detail the capacity of the heart to incorporate adenine, hypoxanthine and ribose into cellular nucleotides. Maximum rates of catalysis by enzymes on the salvage pathways have been established. Whilst the rate of incorporation of adenine into the ATP pool appears to depend upon intracellular concentrations of adenine and phosphoribosylpyrophosphate, for hypoxanthine the pattern is more complex. Hypoxanthine is salvaged at a slow rate compared with adenine, and is incorporated into GTP and IMP as well as into adenine nucleotides. The rate of incorporation of hypoxanthine into both IMP and ATP is accelerated in myocytes incubated with ribose. However, the rate-limiting reaction appears to be that catalysed by adenylosuccinate synthetase, for the rate of ATP synthesis is not accelerated when hypoxanthine concentration is increased from 10 to 50 microM, while the rate of IMP synthesis is more than doubled. Adenine and hypoxanthine phosphoribosyl transferases are present in equal catalytic amounts, but rat cardiac myocytes have very little adenylosuccinate synthetase activity. Exogenous ribose is incorporated into adenine nucleotides in amounts equimolar with adenine or hypoxanthine.     

Elevation of cAMP in cultured mesangial cells diminishes vasopressin-stimulated increases of phosphate uptake and 32P-specific activity in ATP but has no effect on phosphoinositide metabolism

Agents known to elevate intracellular cyclic AMP (cAMP) in cultured mesangial cells (e.g., isoproterenol with and without isobutylmethylxanthine (MIX] inhibit vasopressin-induced contraction. Since contraction of these cells in response to vasopressin is accompanied by release of inositol trisphosphate and increased intracellular ionized calcium, we wanted to determine whether cAMP is exerting its relaxing effect by altering phosphoinositide metabolism. Isoproterenol and MIX did not diminish the release of inositol trisphosphate in response to vasopressin. However, the stimulated 32P incorporation into phospholipids seen with vasopressin treatment was diminished by prior treatment with isoproterenol-MIX. Since incorporation of 32P into phospholipids is not only dependent on phospholipid synthesis but also on the amount of label in the gamma-phosphate of ATP, we determined the specific activity of 32P in ATP. We found that suppression of 32P incorporation into phospholipids in cells treated with isoproterenol-MIX was paralleled by a decline of specific activity of 32P in ATP. Furthermore, the changes in ATP specific activity were paralleled by similar changes in phosphate uptake into the cells. Thus, diminished phosphate uptake (transport) could account for the decline of 32P content in phospholipids and ATP following treatment of mesangial cells with isoproterenol-MIX.     

THE EFFECT OF EXTRACELLULAR POTASSIUM CONCENTRATION ON PROTEIN SYNTHESIS IN GUINEA-PIG HIPPOCAMPAL SLICES1,2

Abstract— We have measured the effect of small variations in extracellular potassium concentrations ([K⁺]) upon the incorporation of radioactively labelled amino acid into the protein of the isolated guinea-pig hippocampal slice. The slice is super-perfused with glucose fortified buffer and maintains an ATP concentration of 33–36 nmol/mg protein and incorporates lysine into protein at a rate of 0.82 pmol/(ig protein/h. Within the range of extracellular K⁺ from 1.3 to 8.1 mil the change in the rate of lysine incorporation into protein is proportional to the logarithm of the extracellular K⁺ concentration. Incorporation increases by about 100% over this range. Measurements of the specific activity of the presumed intracellular amino acid pool indicate that the effect of changes in extracellular [K⁺] is to alter the rate of protein synthesis rather than alter the availability of radioactively labelled precursor. Altering extracellular [K⁺] does not affect tissue levels of ATP or creatine phosphate, indicating that the effect on amino acid incorporation does not result from an effect upon energy metabolism. It is suggested that this effect of extracellular [K.⁺] may be a means by which changes in cerebral electrical activity lead to changes in the rate of protein synthesis in brain.     

Regulation of proteoglycan synthesis rate in cartilage in vitro: influence of extracellular ionic composition

Load-bearing cartilages regularly experience changes in fluid content as the result of changing load. It has been found that these changes in fluid content influence proteoglycan synthesis. The mechanism for this effect is not known. We have measured the influence of changes in cartilage hydration on the [35S]sulphate incorporation rate in both bovine nasal and human articular cartilage in medium whose concentration varied over the range 0.2-2-times physiological strength. In physiological medium the incorporation rate fell in proportion to fluid loss with a 10% fall in cartilage hydration resulting in a 30-50% decrease in 35S-incorporation rates. However, in medium of 0.5-times physiological strength, where the incorporation rate was only 40% of control values, the incorporation rate increased initially rather than falling as the cartilage lost fluid. These changes in hydration and hence proteoglycan content resulted in changes in the extracellular ionic composition of cartilage. When this was monitored in terms of [Na+]c, the internal sodium concentration, as a marker for changes in cartilage ionic composition, we found that incorporation rate varied with [Na+]c rather than directly with hydration.     

Changes in intracellular cations during the cell cycle in HeLa cells

Intracellular Na+, K+, and Mg2+ concentrations have been measured during the HeLa cell cycle and compared with changes in oxygen utilization and macromolecular synthesis. Cell water content remains relatively constant at 79 +/- 1% during the cell cycle. A biphasic change in intracellular Na+ occurs with low values as cells reach peak S phase and again in early G1. The decrease in S coincides with an increase in cell volume during increased macromolecular synthesis. The fall in intracellular Na+ during mitosis/early G1 coincides with decreased energy utilization as macromolecular synthesis decreases with a continued decrease in [Na+]i in G1 corresponding to a period of increasing cell volume and an increase in protein synthesis. Intracellular Na+ is relatively high during late S/G2 when phosphate incorporation into protein and phospholipid is maximal. Intracellular K+ concentrations largely parallel intracellular Na+ levels although the intracellular K+:Na+ ratio is significantly lower as the cell volume increases during late G2/mitosis. Additions of a Na+-pump inhibitor (strophanthidin) not only caused a rise in [Na+]i and fall in [K+]i but also inhibited protein synthesis. Conversely, addition of a protein synthesis inhibitor (cycloheximide) blocked amino acid incorporation and produces a fall in intracellular Na+ levels. These findings indicate that intracellular Na+ and K+ play an important role in regulating cell hydration during the cell cycle and that changes in Na+, K+-ATPase activity, synthesis and/or utilization of high energy phosphate compounds, fluid phase turnover (endocytosis), Na+:H+ exchange (pHi), Donnan forces, and ionic adsorption may all be involved.     

Enhanced channelling of sulphate through a rapidly exchangeable sulphate pool in response to stimulated glycosaminoglycan synthesis in pancreatic epithelial cells.

The ability of cells to decorate glycosaminoglycans (GAGs) with sulphate in highly specific patterns is important to extracellular matrix biogenesis and placing appropriate glycosulphated ligands on the cell surface. We have examined sulphate metabolism in two pancreatic duct epithelial cell lines - PANC-1 and CFPAC-1 (derived from a cystic fibrosis patient) with a view to understanding how pancreatic cells utilise intracellular sulphate. [35S]Sulphate uptake was rapid and reached near steady state levels within 10 min. However, the intracellular specific activity of [35S]sulphate for PANC-1 and CFPAC-1 reached only 35 and 10%, respectively, of the medium specific activity at 10 min. Therefore, sulphate appears to reside within two compartments; a rapidly exchangeable sulphate pool (RESP) and a slowly exchangeable sulphate pool (SESP). Reducing chloride in the medium, increased the specific activity of [35S]sulphate within cells and increased the size of the inorganic sulphate pool, suggesting that the RESP was enlarged. Sulphate pools were not different in size between the two cell lines in physiological NaCl. Increasing the size of the sulphate pool had no effect on [35S]sulphate:[3H]glucosamine ratios incorporated into glycosaminoglycans (GAGs); however, stimulating the synthesis of GAGs with 4-methylumbelliferyl-beta-d-xyloside, stably elevated [35S]:[3H] ratios. This was due to higher [35S]sulphate incorporation. [35S]Cysteine contributed less than 0.1% of the cells' sulphate requirements. We conclude that in the face of elevated demand for sulphate, pancreatic cells appear to channel a greater proportion through the RESP.     

Studies on the incorporation of [U-14C]glucose and [35S]sulphate into the acid glycosaminoglycans of neonatal rat skin

1. The incorporation of [(35)S]sulphate in vivo into the acid-soluble intermediates extracted from young rat skin showed three sulphated hexosamine-containing components. 2. The rates of synthesis of these components were determined in vivo by measuring the incorporation of radioactivity from [U-(14)C]glucose into their isolated hexosamine moieties. 3. The incorporation of radioactivity from [U-(14)C]glucose into the isolated hexosamine and uronic acid moieties of the acid glycosaminoglycans was also measured. These results, combined with those obtained on the intermediary pathways of hexosamine and uronic acid biosynthesis previously determined in this tissue, indicated that the acid-soluble sulphated hexosamine-containing components were not precursors of the sulphated hexosamine found in the acid glycosaminoglycans. 4. The rates of synthesis of the acid glycosaminoglycan fractions were calculated from the incorporation of radioactivity from [U-(14)C]glucose into the hexosamine moiety. The sulphated components containing principally dermatan sulphate, chondroitin 6-sulphate and in smaller amounts, chondroitin 4-sulphate, heparan sulphate and heparin appeared to be turning over about twice as rapidly as hyaluronic acid and about four times as rapidly as the small keratan sulphate fraction. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from the incorporation of [(35)S]sulphate and were in agreement with those from (14)C-labelling studies.     

Role of the adenylate system and glycolytic flux in the control of protein synthesis in isolated rat lung cells

(1) Glucose stimulates the incorporation of amino acids into protein in lung cells isolated by digestion of the lung stroma with collagenase. This effect reflects mainly an increase in protein synthesis since no effect of glucose had been found to the uptake of amino acid precursors and, although glucose decreases the rate of intracellular proteolysis by 15%, this effect cannot account for the increased incorporation of radioactivity into proteins. Furthermore, glucose did not induce any significant change in the intracellular content of valine. (2) For glucose to act on protein synthesis, it must be glycolyzed since its stereoisomer, L-glucose, which is not metabolized by lung cells, has no effect. (3) The mechanism of glucose action does not seem to be related simply to variations of cellular ATP content or energy charge. The following arguments seem to support this conclusion: (i) glucose does not bring about significant variations in the concentration of reactants of the adenylate system; (ii) the increase in protein synthesis induced by glucose in energy-depleted cells correlates with a rise in ATP content and energy charge; however, adenosine, which increases ATP levels in a form quantitatively similar to glucose, is unable to affect protein synthesis: (iii) glucose also accelerates the incorporation of amino acids into proteins in adenosine-treated lung cells in which the ATP concentration was almost double that of the control and the energy charge was considerably elevated, ruling out the possibility that a rise in the steady-state concentration of ATP and/or energy charge alone could be responsible for the acceleration of protein synthesis. (4) It can be concluded that the effect of glucose in increasing protein synthesis in lung cells is dependent on some signal arising from its breakdown and not to variations in the concentration of reactants or energy charge of the adenylate system.     

ATP generation in the Trypanosoma brucei procyclic form: cytosolic substrate level is essential, but not oxidative phosphorylation

Trypanosoma brucei is a parasitic protist responsible for sleeping sickness in humans. The procyclic form of this parasite, transmitted by tsetse flies, is considered to be dependent on oxidative phosphorylation for ATP production. Indeed, its respiration was 55% inhibited by oligomycin, which is the most specific inhibitor of the mitochondrial F0/F1-ATP synthase. However, a 10-fold excess of this compound did not significantly affect the intracellular ATP concentration and the doubling time of the parasite was only 1.5-fold increased, suggesting that oxidative phosphorylation is not essential for procyclic trypanosomes. To further investigate the sites of ATP production, we studied the role of two ATP producing enzymes, which are involved in the synthesis of pyruvate from phosphoenolpyruvate: the glycosomal pyruvate phosphate dikinase (PPDK) and the cytosolic pyruvate kinase (PYK). The parasite was not affected by PPDK gene knockout. In contrast, inhibition of PYK expression by RNA interference was lethal for these cells. In the absence of PYK activity, the intracellular ATP concentration was reduced by up to 2.3-fold, whereas the intracellular pyruvate concentration was not reduced. Furthermore, we show that this mutant cell line still excreted acetate from d-glucose metabolism, and both the wild type and mutant cell lines consumed pyruvate present in the growth medium with similar high rates, indicating that in the absence of PYK activity pyruvate is still present in the trypanosomes. We conclude that PYK is essential because of its ATP production, which implies that the cytosolic substrate level phosphorylation is essential for the growth of procyclic trypanosomes.     

Lipid biosynthesis in liver slices of the foetal guinea pig.

Lipid synthesis as measured by the incorporation of acetate or 3H2O into slices of foetal liver, is much higher than in slices of adult liver and shows a peak at about two-thirds of gestation. At this time the synthesis from glucose was low and reached a peak 10 days later. The changes in the activity of ATP citrate lyase, which mirrored acetate incorporation, and the effect of glucose and pyruvate on acetate corporation into lipid suggests that some of the lipid synthesis occurs via intramitochondrial acetyl-CoA production from acetate. Despite this, lipid synthesis was not inhibited by (-)-hydroxycitrate. The low rate of synthesis from glucose at two-thirds of gestation is ascribed to the low activity of pyruvate carboxylase at this time and a role for a phosphoenolpyruvate carboxykinase in providing oxaloacetate for lipogenesis is proposed. The activity of fatty acid synthetase broadly agreed with the changes in lipid synthesis, whereas the activity of acetyl-CoA carboxylase was barely sufficient to account for the rates of lipid synthesis in vivo. Acetate and short-chain fatty acids are likely to be the major precursors for lipid synthesis in vivo.     

The effects of cycloheximide on the biosynthesis and secretion of proteoglycans by chondrocytes in culture.

Proteoglycans synthesized by rat chondrosarcoma cells in culture are secreted into the culture medium through a pericellular matrix. The appearance of [35S]sulphate in secreted proteoglycan after a 5 min pulse was rapid (half-time, t 1/2 less than 10 min), but that of [3H]serine into proteoglycan measured after a 15 min pulse was much slower (t 1/2 120 min). The incorporation of [3H]serine into secreted protein was immediately inhibited by 1 mM-cycloheximide, but the incorporation of [35S]sulphate into proteoglycans was only inhibited gradually(t 1/2 79 min), suggesting the presence of a large intracellular pool of proteoglycan that did not carry sulphated glycosaminoglycans. Cultures were pulsed with [3H]serine and [35S]sulphate and chased for up to 6 h in the presence of 1 mM-cycloheximide. Analysis showed that cycloheximide-chased cells secreted less than 50% of the [3H]serine in proteoglycan of control cultures and the rate of incorporation into secreted proteoglycan was decreased (from t 1/2 120 min to t 1/2 80 min). Under these conditions cycloheximide interfered with the flow of proteoglycan protein core along the route of intracellular synthesis leading to secretion, as well as inhibiting further protein core synthesis. The results suggested that the newly synthesized protein core of proteoglycan passes through an intracellular pool for about 70-90 min before the chondroitin sulphate chains are synthesized on it, and it is then rapidly secreted from the cell. Proteoglycan produced by cultures incubated in the presence of cycloheximide and labelled with [35S]sulphate showed an increase with time of both the average proteoglycan size and the length of the constituent chondroitin sulphate chain. However, the proportion of synthesized proteoglycans able to form stable aggregates did not alter.     

Studies on insulin-stimulated phosphorylation of acetyl-CoA carboxylase, ATP citrate lyase and other proteins in rat epididymal adipose tissue. Evidence for activation of a cyclic AMP-independent protein kinase.

Protein kinase activity in high-speed supernatant fractions prepared from rat epididymal adipose tissue previously incubated in the absence or presence of insulin was investigated by following the incorporation of 32P from [gamma-32P]ATP into phosphoproteins separated by sodium dodecyl sulphate/polyacrylamide-gel electro-phoresis. Incorporation of 32P into several endogenous proteins in the supernatant fractions from insulin-treated tissue was significantly increased. These included acetyl-CoA carboxylase and ATP citrate lyase (which exhibit increased phosphorylation within fat-cells exposed to insulin), together with two unknown proteins of subunit Mr 78000 and 43000. The protein kinase activity increased by insulin was distinct from cyclic AMP-dependent protein kinase, was not dependent on Ca2+ and was not appreciably affected by dialysis or gel filtration. The rate of phosphorylation of added purified fat-cell acetyl-CoA carboxylase and ATP citrate lyase was also increased by 60-90% in high-speed-supernatant fractions prepared from insulin-treated tissue. No evidence for any persistent changes in phosphoprotein phosphatase activity was found. It is concluded that insulin action on acetyl-CoA carboxylase, ATP citrate lyase and other intracellular proteins exhibiting increased phosphorylation involves an increase in cyclic AMP-independent protein kinase activity in the cytoplasm. The possibility that the increase reflects translocation from the plasma membrane, perhaps after phosphorylation by the protein tyrosine kinase associated with insulin receptors, is discussed.     

Reduced contribution from Na+/H+ exchange to acid extrusion during anoxia in adult rat hippocampal CA1 neurons

The effect of anoxia on Na+/H+ exchange activity was examined in acutely isolated adult rat hippocampal CA1 neurons loaded with the H+-sensitive fluorophore, BCECF. Five-minute anoxia imposed under nominally HCO3-/CO2-free conditions induced a fall in pHi, the magnitude of which was smaller following prolonged exposure to medium in which N-methyl-D-glucamine (NMDG+) was employed as an extracellular Na+ (Na(+)(o)) substitute. Also consistent with the possibility that Na+/H+ exchange becomes inhibited soon after the induction of anoxia, rates of Na(+)(o)-dependent pHi recovery from internal acid loads imposed during anoxia were slowed, compared to rates of Na(+)(o)-dependent pHi recovery observed prior to anoxia. At the time at which rates of pHi recovery were reduced during anoxia, cellular adenosine triphosphate (ATP) levels had fallen to 35% of preanoxic levels, suggesting that ATP depletion might contribute to the observed inhibition of Na+/H+ exchange. In support, incubation of neurons with 2-deoxyglucose and antimycin A under normoxic conditions induced a fall in cellular ATP levels that was also associated with reduced Na(+)(o)-dependent rates of pHi recovery from imposed acid loads; conversely, pre-treatment with 10 mm creatine attenuated the effects of anoxia to reduce both ATP levels and Na(+)(o)-dependent rates of pHi recovery from internal acid loads. Taken together, the results are consistent with the possibility that functional Na+/H+ exchange activity in adult rat CA1 neurons declines soon after the onset of anoxia, possibly as a result of anoxia-induced falls in intracellular ATP.     

Kinetic control of mitochondrial ATP synthesis

In order to gain a clearer understanding of the kinetic control of ATP synthesis, rat liver and rat heart mitochondria were incubated under conditions that resulted in various rates of net ATP synthesis or ATP hydrolysis. Radiolabeled phosphate was included in the incubation media, and exchange rates between phosphate and ATP were determined as a function of rates of net ATP synthesis. Since ATP synthase is a highly reversible enzyme, the catalyzed reaction was expected to approach equilibrium especially at low rates of respiration and net ATP synthesis. Thus ADP + Pi V1 in equilibrium V2 ATP. If V1 is the rate of incorporation of radiolabeled phosphate into ATP, then net ATP synthesis (or hydrolysis) is V1 - V2. Since V1 and V1 - V2 could be measured, it was possible to calculate V2. V1 doubled in the transition from zero to maximal net ATP synthesis, whereas V2 decreased by over 90% when the rate of ATP synthesis was high due to high-media ADP. In heart mitochondria at 37 degrees C when respiration increased from 104 +/- 10 to 842 +/- 51 nanoatoms of O2/(min X mg), incorporation of [33P]phosphate into ATP (V1) increased from 1,100 +/- 60 to 1,978 +/- 121 and V2 decreased from 1,100 to near zero. These data demonstrate that mitochondrial ATP synthesis does not occur near equilibrium under physiological conditions and relatively high rates of ATP synthesis. A reaction with a high ratio of forward to reverse flux is obviously not near equilibrium. The important most sensitively controlled reaction appears to be V2, ATP hydrolysis. Possible mechanisms of kinetic control of V2 are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

The lack of effect of glucosamine sulphate on aggrecan mRNA expression and (35)S-sulphate incorporation in bovine primary chondrocytes

Glucosamine and glucosamine sulphate have been promoted as a disease-modifying agent to improve the clinical symptoms of osteoarthritis. The precise mechanism of the action of the suggested positive effect of glucosamine or glucosamine sulphate on cartilage proteoglycans is not known, since the level of glucosamine in plasma remains very low after oral administration of glucosamine sulphate. We examined whether exogenous hexosamines or their sulphated forms would increase steady-state levels of aggrecan and hyaluronan synthase (HAS) or glycosaminoglycan synthesis using Northern blot and (35)S-sulphate incorporation analyses. Total RNA was extracted from bovine primary chondrocytes which were cultured either in 1 mM concentration of glucosamine, galactosamine, mannosamine, glucosamine 3-sulphate, glucosamine 6-sulphate or galactosamine 6-sulphate for 0, 4, 8 and 24 h, or in three different concentrations (control, 100 microM and 1 mM) of glucosamine sulphate salt or glucose for 24 or 72 h. Northern blot assay showed that neither hexosamines nor glucosamine sulphate salt stimulated aggrecan and HAS-2 mRNA expression. Glycosaminoglycan synthesis remained at a control level in the treated cultures, with the exception of mannosamine which inhibited (35)S-sulphate incorporation in low-glucose DMEM treatment. In our culture conditions, hexosamines or their sulphated forms did not increase aggrecan expression or (35)S-sulphate incorporation.     

Regulation of heparan sulphate metabolism by adenosine 3′:5′-cyclic monophosphate in hepatocytes in culture

Freshly isolated rat hepatocytes maintained as monolayers in a serum-free medium synthesize sulphated glycosaminoglycans, most of which behave as heparan sulphate and are mainly distributed into intracellular compartments. Cyclic AMP, dibutyryl cyclic AMP, glucagon, noradrenaline, prostaglandin E(1), and theophylline, all drugs and hormones known to increase intracellular cyclic AMP concentrations, decreased the incorporation of (35)SO(4) (2-) into heparan sulphate of intra-, extra- and peri-cellular pools. The inhibition mediated by dibutyryl cyclic AMP was dose-dependent and observed as early as 2h after exposure to the drug. In the presence of 1mm-dibutyryl cyclic AMP, incorporation of (35)SO(4) (2-) or [(14)C]glucosamine into heparan sulphate was decreased to 40-50%, suggesting that dibutyryl cyclic AMP interfered with the synthesis of heparan sulphate. This was further supported by pulse-chase experiments, where dibutyryl cyclic AMP had no effect on the degradation of sulphated glycosaminoglycans. Heparan sulphates synthesized and secreted into the extracellular pool in the presence of dibutyryl cyclic AMP were smaller in size, whereas the degree of sulphation and molecular size of the heparan sulphate chains released by beta-elimination from these proteoglycans were not different from control values. In the presence of 1mm-cycloheximide, (35)SO(4) (2-) incorporation was decreased to 5%. Addition of p-nitrophenyl beta-d-xyloside, an artificial acceptor of glycosaminoglycan chain synthesis, enhanced this incorporation to 18%. Dibutyryl cyclic AMP did not have any inhibitory effect on the synthesis of chains initiated on p-nitrophenyl beta-d-xylosides. Incorporation of [(3)H]serine into heparan sulphate was not affected by dibutyryl cyclic AMP, whereas the degree of substitution of serine residues with heparan sulphate chains was less in heparan sulphate synthesized in the presence of dibutyryl cyclic AMP, suggesting that cyclic AMP exerts its effect on the metabolism of sulphated glycosaminoglycans by affecting the transfer of xylose on to the protein core.     

Acyl-CoAs are functionally channeled in liver: potential role of acyl-CoA synthetase

Acyl-CoA synthetase (ACS) catalyzes the activation of long-chain fatty acids to acyl-CoAs, which can be metabolized to form CO(2), triacylglycerol (TAG), phospholipids (PL), and cholesteryl esters (CE). To determine whether inhibiting ACS affects these pathways differently, we incubated rat hepatocytes with [(14)C]oleate and the ACS inhibitor triacsin C. Triacsin inhibited TAG synthesis 70% in hepatocytes from fed rats and 40% in starved rats, but it had little effect on oleate incorporation into CE, PL, or beta-oxidation end products. Triacsin blocked [(3)H]glycerol incorporation into TAG and PL 33 and 25% more than it blocked [(14)C]oleate incorporation, suggesting greater inhibition of de novo TAG synthesis than reacylation. Triacsin did not affect oxidation of prelabeled intracellular lipid. ACS1 protein was abundant in liver microsomes but virtually undetectable in mitochondria. Refeeding increased microsomal ACS1 protein 89% but did not affect specific activity. Triacsin inhibited ACS specific activity in microsomes more from fed than from starved rats. These data suggest that ACS isozymes may be functionally linked to specific metabolic pathways and that ACS1 is not associated with beta-oxidation in liver.     

The lack of effect of glucosamine sulphate on aggrecan mRNA expression and 35S-sulphate incorporation in bovine primary chondrocytes

Glucosamine and glucosamine sulphate have been promoted as a disease-modifying agent to improve the clinical symptoms of osteoarthritis. The precise mechanism of the action of the suggested positive effect of glucosamine or glucosamine sulphate on cartilage proteoglycans is not known, since the level of glucosamine in plasma remains very low after oral administration of glucosamine sulphate. We examined whether exogenous hexosamines or their sulphated forms would increase steady-state levels of aggrecan and hyaluronan synthase (HAS) or glycosaminoglycan synthesis using Northern blot and ³⁵S-sulphate incorporation analyses. Total RNA was extracted from bovine primary chondrocytes which were cultured either in 1 mM concentration of glucosamine, galactosamine, mannosamine, glucosamine 3-sulphate, glucosamine 6-sulphate or galactosamine 6-sulphate for 0, 4, 8 and 24 h, or in three different concentrations (control, 100 μM and 1 mM) of glucosamine sulphate salt or glucose for 24 or 72 h. Northern blot assay showed that neither hexosamines nor glucosamine sulphate salt stimulated aggrecan and HAS-2 mRNA expression. Glycosaminoglycan synthesis remained at a control level in the treated cultures, with the exception of mannosamine which inhibited ³⁵S-sulphate incorporation in low-glucose DMEM treatment. In our culture conditions, hexosamines or their sulphated forms did not increase aggrecan expression or ³⁵S-sulphate incorporation.