Human glioma U-251 cells contain type 1 plasminogen activator inhibitor in a rapidly releasable form

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Because recent information suggests that the localized deposition of protease inhibitors is one mechanism by which cells regulate pericellular proteolysis during tissue invasion, the distribution of type 1 plasminogen activator inhibitor (PA1-1) associated with the invasive human glioma cell line U-251 was investigated. Direct and reverse fibrin zymography indicated the presence of urokinase-like plasminogen activator (u-PA) and PAI-1 in U-251 conditioned media and cell lysates. PA1-1 antigen was detected immunologically in cytoplasmic granules present within cellular processes of U-251 cells and these organelles could be isolated on Percoll density gradients in a high density band. In contrast, u-PA activity and another secreted protein, amyloid beta-protein precursor, were only present in the low density region of the gradients. Functional analysis of PAI-1 in the granules contained within the high density fractions revealed the presence of active PAI-1. Incubation of U-251 cells with the secretagogue, 8-bromoadenosine 3':5'-cyclic monophosphate, resulted in a 3-fold increase in the release of PAI-1 in the media conditioned by these cells. These data suggest that the human glioma cell line U-251 contains PAI-1 in a rapidly releasable form, which may provide another mechanism by which these tumors could regulate proteolytic activity in a localized manner.