Tissue plasminogen activator (tPA) has been implicated in a variety of important cellular functions, including learning-related synaptic plasticity and potentiating N-methyl-D-aspartate (NMDA) receptor-dependent signaling. These findings suggest that tPA may localize to, and undergo activity-dependent secretion from, synapses; however, conclusive data supporting these hypotheses have remained elusive. To elucidate these issues, we studied the distribution, dynamics, and depolarization-induced secretion of tPA in hippocampal neurons, using fluorescent chimeras of tPA. We found that tPA resides in dense-core granules (DCGs) that traffic to postsynaptic dendritic spines and that can remain in spines for extended periods. We also found that depolarization induced by high potassium levels elicits a slow, partial exocytotic release of tPA from DCGs in spines that is dependent on extracellular Ca(+2) concentrations. This slow, partial release demonstrates that exocytosis occurs via a mechanism, such as fuse-pinch-linger, that allows partial release and reuse of DCG cargo and suggests a mechanism that hippocampal neurons may rely upon to avoid depleting tPA at active synapses. Our results also demonstrate release of tPA at a site that facilitates interaction with NMDA-type glutamate receptors, and they provide direct confirmation of fundamental hypotheses about tPA localization and release that bear on its neuromodulatory functions, for example, in learning and memory.

SummaryStarvation of Leishmania donovani parasites for purines leads to a rapid amplification in purine nucleobase and nucleoside transport. Studies with nucleoside transport-deficient L. donovani indicate that this phenomenon is mediated by the nucleoside transporters LdNT1 and LdNT2, as well as by the purine nucleobase transporter LdNT3. The escalation in nucleoside transport cannot be ascribed to an increase in either LdNT1 or LdNT2 mRNA. However, Western analyses on parasites expressing epitope-tagged LdNT2 revealed a marked upregulation in transporter protein at the cell surface. Kinetic investigations of LdNT1 and LdNT2 activities from purine-replete and purinestarved cells indicated that both transporters exhibited significant increases in V max for their ligands under conditions of purine-depletion, although neither transporter displayed an altered affinity for its respective ligands. Concomitant with the increase in purine nucleoside and nucleobase transport, the purine salvage enzymes HGPRT, XPRT and APRT were also upregulated, suggesting that under conditions where purines are limiting, Leishmania parasites remodel their purine metabolic pathway to maximize salvage. Moreover, qRT-PCR analyses coupled with cycloheximide inhibition studies suggest that the underlying molecular mechanism for this augmentation in purine salvage occurs post-transcriptionally and is reliant on de novo protein synthesis.

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