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OBJECTIVE: The efficacy of glucosamine sulfate (GS) in the symptomatic treatment of patients with osteoarthritis (OA) is suggested to be mediated by still unknown effects on the altered OA cartilage. DESIGN: Using human OA chondrocytes in culture, the effects of GS on protein synthesis, caseinase, collagenase, phospholipase A2 (PLA2) and protein kinase C (PKC) activities as well as production of nitric oxide and cyclic AMP were studied in both cells and culture medium. RESULTS: GS significantly reduced PLA2 activity, and more modestly collagenase activity, in the OA chondrocytes in a dose-dependent manner. By contrast, PLA2 and collagenase activity of the culture medium was not modified. No effects on caseinase activity was seen. GS significantly and dose-dependently increased protein synthesis. GS did not modify nitric oxide and cAMP production but significantly increased PKC production. CONCLUSION: GS modified cultured OA chondrocyte metabolism by acting on PKC, cellular PLA2, protein synthesis and possibly collagenase activation. Extrapolation of the effect to the in-vivo situation remains hypothetical but they might represent some possible mechanisms of action of the drug in human.

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STUDY DESIGN: Prospective study of phospholipase A2 activity in the serum and intervertebral discs of patients undergoing surgery for sciatica due to disc herniation. OBJECTIVES: To determine correlations between herniated disc phospholipase A2 and clinical, radiographic, and anatomic signs of common sciatica; to evaluate serum phospholipase A2 activity as a marker of disc phospholipase A2; and to investigate the in vivo effect of piroxicam on disc phospholipase A2. SUMMARY OF BACKGROUND DATA: Several studies suggest disc inflammation as a mechanism of sciatica due to disc herniation, and phospholipase A2 emerges as a key enzyme of cartilage and disc tissues. METHODS: Phospholipase A2 activity was determined, using the degradation of a specific substrate, in the serum and discs of 31 patients (14 treated with acetaminophen and 17 treated with piroxicam) undergoing surgery for sciatica due to lumbar disc herniation. Visual analog scale for pain, Dallas Pain Questionnaire, Lasègue's sign, radiographic stage of degeneration of the herniated disc, volume of disc herniation shown by computed tomography, and surgical findings were recorded. RESULTS: Disc phospholipase A2 activity was independent of the patient's age or sex, the radiologic stage of disc degeneration, and the volume of the herniation, and showed no significant correlation with Lasègue's sign or pain measured on a visual analog scale. The correlation between disc phospholipase A2 and the Dallas category of items measuring the impact of pain on daily activities approached the level of significance (P = 0.07). Disc phospholipase A2 activity was significantly higher in cases of sequestrated discs than in other herniations. Disc phospholipase A2 was significantly correlated with serum phospholipase A2, and was significantly lower in patients treated with piroxicam than in those treated with acetaminophen. CONCLUSIONS: Disc phospholipase A2 is thought to participate in the physiopathology of sciatica and to bemodulated by nonsteroidal anti-inflammatory drug therapy. Serum phospholipase A2 is suggested as a biologic marker of disc inflammation in patients with sciatica.

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Calcitonin (CT) is a known potent inhibitor of bone resorption but its effect on cartilage enzymatic degradation has been incompletely studied. Salmon CT, at a concentration of 0, 0.1, 0.25, 0.5, 2.5 and 50 ng/ml, was added at 24 or 72 h to the culture medium of chondrocytes from human osteoarthritic hips and knees. The spontaneous collagenolytic activity, measured using a radiolabeled type II collagen, was inhibited by CT in a dose-dependent manner. However, CT had no effect on the total collagenolytic activity assayed after APMA activation. Stromelysin and plasmin activity, measured by degradation of casein and a synthetic substrate, were also unaffected by CT. Chondrocyte phospholipase A2 activity, assayed using a labeled specific substrate, was decreased by CT. Chondrocyte pre-incubation with CT significantly decreased the cell binding of labeled TNF alpha, but did not affect IL-1 beta cell binding. Attachment of chondrocytes on fibronectin was markedly stimulated by CT, while attachment to type II collagen was not. Significant effects were obtained using at least 2 or 5 ng/ml of CT. CT appears to decrease collagenolytic activity by decreasing its activation and/or increasing its inhibition by tissue inhibitors of metalloproteinases (TIMP). CT might act on osteoarthritic chondrocyte activation via mechanisms such as phospholipase A2 activity, human necrosis factor-alpha or fibronectin receptor expression.

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The effect of non-steroidal anti-inflammatory drugs (NSAIDs) on the activity of glycosyltransferases required for the synthesis of the polysaccharide chains of proteoglycans, was studied in human osteoarthritic cartilage in vitro. Using exogenous acceptors, salicylate and indomethacin suppressed the activity of glucuronyl- and xylosyltransferases in a concentration-dependent manner, but had little effect on N-acetylgalactosaminyl- and galactosyltransferases. When used at a concentration derived from the values found in the synovial fluid, salicylate, indomethacin and chloroquine significantly suppressed the activity of glucuronyl- and xylosyltransferases, while tiaprofenic acid, paracetamol (acetaminophen), floctafenine, ketoprofen, ibuprofen and tenoxicam had no effect on the enzymes. An alteration of some glycosyltransferases could explain the reported suppressive effect of some NSAIDs on cartilage proteoglycan synthesis.

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Chondrocyte metalloproteinases appear to play a major role in the development of osteoarthritis. The intracellular post-traductional mechanisms regulating collagenase and proteoglycanase are not known. Calmodulin antagonists including phenothiazine and sulfonamide derivatives significantly increased proteoglycanase activity and decreased collagenase activity. H-7, a specific inhibitor of protein kinase C, had no effect on the two metalloproteinase activities, and calmodulin was ineffective in in vitro assays upon metalloproteinase activities. We postulate that collagenase and proteoglycanase activities are controlled by calmodulin-dependent regulation.

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We have previously described several receptors on the chondrocyte membrane. In an attempt to further characterize the coupling mechanisms of serotoninergic receptors, here we examined the involvement of serotonin in the phospholipase A2 activity. Serotonin dose-dependently stimulated phospholipase A2. This activation enhanced collagenase type II activity and had no effect on proteoglycanase activity.

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Six glycosyltransferases (mannosyl-, glucosyl-, N-acetyl-glucosaminyl-, galactosyl-, sialyl- and fucosyltransferases) are studied and characterized for their optimal conditions and their relations with interfering reactions (glycosyl-nucleotide pyrophosphatases, glycosidases and proteinases) in chondrocytes from osteoarthritic and normal human articular cartilage. Osteoarthritis induces increased activities for five glycosyl-transferases. The observed modifications are not explained by alterations in physico-chemical parameters of the enzymes or by intervention of glycosyl-nucleotide pyrophosphatases, glycosidases or proteolytic enzymes.

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The tricyclic antidepressant desipramine, when added to culture medium, gave rise in C6 rat glioma cells to a decrease of the activity of the enzyme asialofetuin sialyltransferase. The inhibition was dose and time dependent and was observed in both multiplying cells and cells blocked with 2 mM thymidine or depletion of amino acids. This inhibition was rather specific to the sialyltransferase, as under the conditions where this enzyme was inhibited up to 70%, other enzymes such as dolichol phosphate mannose synthetase, glutamine synthetase, and glycerol phosphate dehydrogenase remained unaffected. This inhibition was not reversed after removal of desipramine from the medium and was not observed by direct addition of desipramine to the sialyltransferase incubation assay. Under the same conditions, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], which is known to be a potent calmodulin antagonist and an inhibitor of calmodulin-dependent kinases, gave the same concentration-dependent inhibition profile of sialyltransferase as desipramine, whereas H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], which is an inhibitor of protein kinase C and cyclic nucleotide-dependent kinases, had no effect. So, it is suggested that desipramine inhibits the sialyltransferase activity in C6 glioma cells through a calmodulin-dependent system.

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Circadian variations of the acetylcholine muscarinic receptor and some glycosyltransferases were studied in brain using multivariate analysis. Highly significant correlations exist between fucosyltransferase, sialyltransferase and galactosyltransferase and to a lesser extent between both of these enzymes and acetylcholine receptor. No correlation appeared between these enzymes and dolichol phosphate mannose synthase.

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GDP-fucose: asialofetuin fucosyltransferase from sheep brain was fractionated on a sucrose gradient into two activity peaks. Using purification on Ficoll adapted from the proposed method [(1980) J. Neurochem. 35, 281-296], double localisation of cerebral fucosyltransferase was confirmed and the subcellular active fractions identified as light microsomes and mitochondria.

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