The relationship between proteoglycan synthesis in Swarm chondrocytes and pathways of cellular energy and UDP-sugar metabolism.
The effect of anaerobic culture conditions and various metabolic inhibitors on 35S-proteoglycan synthesis, UDP-sugar pools, and the ATP pool were investigated in confluent, primary, day 1 cultures of Swarm chondrosarcoma chondrocytes. (i) Incubation under a nitrogen atmosphere for 6 h did not affect 35S-proteoglycan synthesis or the pool size for UDP-glucuronate, other UDP-sugars, or ATP. Incubation with 5 mM sodium azide brought about a 40% reduction of proteoglycan synthesis in the first 30 min but no further change over the subsequent 90 min. UDP-Glucuronate, other UDP-sugar pools, and the ATP level were not affected by azide treatment. The results indicate that proteoglycan synthesis and its energy requirements can be supported entirely by anaerobic metabolism in these cells. (ii) 35S-Proteoglycan synthesis, UDP-sugar production, and nucleotide triphosphate pools were inhibited in a concentration-dependent fashion with sodium iodoacetate. A > 70% reduction of the ATP pool after 30 min treatment suggests that glycolysis is a major target for iodoacetate. Lactate production was inhibited by 40% after 3 h treatment with 10(-4) M iodoacetate. (iii) Glutamine deprivation resulted in a 60% contraction in the UDP-N-acetylhexosamine pool and markedly inhibited 35S-proteoglycan and 3H-protein synthesis. At the same time the UDP-glucose pool expanded to 200%, but the UDP-glucuronate pool was unchanged. The sum of the UDP-N-acetylhexosamine and UDP-hexose pools remained constant. Restoration of glutamine to previously depleted cultures resulted in excessive expansion of the UDP-N-acetylhexosamine pool and excessive contraction of the UDP-hexose pool before both adjusted to normal levels. The UDP-xylose pool was very small. No increases were observed during inhibition of proteoglycan synthesis induced by glutamine deprivation. (iv) 6-Diazo-5-oxo-L-norleucine (DON), a glutamine analogue and amino transferase inhibitor, induced a further contraction of the UDP-N-acetylhexosamine pool and a further decrease in proteoglycan synthesis in glutamine-deprived cultures. Thus cultures use endogenous glutamine during exogenous glutamine deprivation. DON unaccountably stimulated expansion of the UDP-glucuronate pool by 180%, irrespective of whether glutamine was present or not.
Mason
R M
RM
Department of Biochemistry, Charing Cross and Westminster Medical School, London, United Kingdom.
Sweeney
C
C
D053674:Aggrecans; D000409:Alanine / Q000032:analysis; D000693:Anaerobiosis; D000818:Animals; D001386:Azides / Q000494:pharmacology; D002356:Cartilage / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis*; D003980:Diazooxonorleucine / Q000494:pharmacology; D004734:Energy Metabolism; D016326:Extracellular Matrix Proteins; D005973:Glutamine / Q000378:metabolism; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007773:Lactates / Q000032:analysis; D037181:Lectins, C-Type; D011509:Proteoglycans / Q000096:biosynthesis*; D051381:Rats; D019810:Sodium Azide; D014407:Tumor Cells, Cultured; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
C508321:Acan protein, rat; D053674:Aggrecans; D001386:Azides; D011508:Chondroitin Sulfate Proteoglycans; D016326:Extracellular Matrix Proteins; D007461:Iodoacetates; D007773:Lactates; D037181:Lectins, C-Type; D011509:Proteoglycans; D014539:Uridine Diphosphate Sugars; D003980:Diazooxonorleucine; D005973:Glutamine; D019810:Sodium Azide; D000409:Alanine; D019807:Iodoacetic Acid