Neutral amino acid transport in bovine articular chondrocytes

1. The sodium-dependent amino acid transport systems responsible for prolin e, glycine and glutamine transport, together with the sodium-independent sy stems for leucine and tryptophan, have been investigated in isolated bovine chondrocytes by inhibition studies and ion replacement. Each system was ch aracterized kinetically. 2. Transport via system A was identified using the system-specific analogue alpha-methylaminoisobutyric acid (MeAIB) as an inhibitor of proline, glyci ne and glutamine transport. 3. Uptake of proline, glycine and glutamine via system ASC was identified b y inhibition with alanine or serine. 4. System Gly was identified by the inhibition of glycine transport with ex cess sarcosine (a substrate for system Gly) whilst systems A and ASC were i nhibited. This system, having a very limited substrate specificity and tiss ue distribution, was also shown to be Na+ and Cl- dependent. Evidence for e xpression of the system Gly component GLYT-1 was obtained using the reverse transcriptase-polymerase chain reaction (RT-PCR). 5. System N, also of narrow substrate specificity and tissue distribution, was shown to be present in chondrocytes. Na+-dependent glutamine uptake was inhibited by high concentrations of histidine (a substrate of system N) in the presence of excess MeAIB and serine. 6. System L was identified using the system specific analogue 2-aminobicycl o(2,2,1)heptane-2-carboxylic acid (BCH) and D-leucine as inhibitors of leuc ine and tryptophan transport. 7. The presence of system T was tested by using leucine, tryptophan and tyr osine inhibition. It was concluded that this system was absent in the chond rocyte. 8. Kinetic analysis showed the Na+-independent chondrocyte L system to have apparent affinities for leucine and tryptophan of 125 +/- 27 and 36 +/- 11 mu M, respectively. 9. Transport of the essential amino acids leucine and tryptophan into bovin e chondrocytes occurs only by the Na+-independent system L, but with a high er affinity than the conventional L system.