Characterization of sulfate, proline, and glucose transport systems in anterior cruciate and medial collateral ligament cells

Abstract

The present study was undertaken to define the nature of key transport processes for sodium, glucose, proline, and sulfate in primary culture of canine anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cells. Uptake studies using radiolabeled isotopes were performed and Na,K-ATPase activity was determined in cell lysates. At 25 °C both ACL and MCL cells showed a significant uptake of86Rb. Ouabain inhibited Rb uptake by 55% in ACL cells and by 60% in MCL cells. The transport activity of Na,K-ATPase in intact cells was calculated to be 57 and 71 nmol·(mg protein)–1·(15 min)–1, respectively. The enzymatic activity of Na,K-ATPase in cell lysates was observed to be 104 for ACL cells and 121 nmol·(mg protein)–1·(15 min)–1for MCL cells. Cytochalasin B, a known inhibitor of sodium-independent D-glucose transport, completely inhibited D-glucose uptake in ACL and MCL cells. Removal of Na+or addition of 10–5mol/L phlorizin, a potent inhibitor of the sodium-D-glucose cotransporter, did not alter D-glucose uptake, suggesting that glucose entered the cells using a sodium-independent pathway. Both ACL and MCL cells exhibited high sulfate uptake that was not altered by replacement of Na+by N-methyl-D-glucamine, whereas DIDS, an inhibitor of sulfate/anion exchange abolished sulfate uptake in both cell types. Thus, neither cell type seems to possess a sodium-sulfate cotransport system. Rather, sulfate uptake appeared to be mediated by sulfate/anion exchange. Proline was rapidly taken up by ACL and MCL cells and its uptake was reduced by 85% when Na+was replaced by N-methyl-D-glucamine, indicating that proline entered the cells via sodium-dependent cotransport systems. The data demonstrate that both ACL and MCL cells possess a highly active sodium pump, a secondary active sodium-proline cotransport system, and sodium-independent transport systems for D-glucose and sulfate.Key words: ligament, fibroblasts, transport, proline, sulfate, glucose, sodium.