SNARE-dependent glutamate release in megakaryocytes

Objective

The identification of signaling pathways involved in megakaryocytopoiesis is essential for development of novel therapeutics to treat hematological disorders. Following our previous findings that megakaryocytes express functional channel-forming N-methyl-D-aspartate-type glutamate receptors, here we aimed to determine the glutamate release capacity in undifferentiated and differentiated megakaryocytes and the role of soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) proteins that are known to be associated with vesicular exocytosis.

Materials and Methods

Using the megakaryocytic cell line MEG-01, primary megakaryocytes, and tissue sections of bone marrow, reverse transcription polymerase chain reaction, Western blot analysis, and immunolocalization were employed to detect factors required for vesicular glutamate release. Vesicle recycling was monitored by acridine orange and FM1-43 staining and glutamate release activity was assessed by an enzyme-linked fluorimetric assay. Genetically modified MEG-01 cells, with deletion or overexpression of SNARE and vesicular proteins, were also examined for glutamate release activity.

Results

We demonstrated that megakaryocytes express numerous proteins required for vesicular glutamate release, including core SNARE proteins, vesicle-associated membrane protein, soluble N-ethyl maleimide-sensitive factor attachment protein−23, and syntaxin, as well as specific glutamate-loading vesicle proteins, VGLUT1 and VGLUT2. Moreover, active vesicle recycling and differentiation-dependent glutamate release were observed in megakaryocytes. Vesicle-associated membrane protein−deficient MEG-01 cells, which are impaired in vesicle recycling, showed a 30% decrease in released glutamate, whereas overexpression of VGLUT1 exhibited up to a 2.2-fold increase in glutamate release.

Conclusion

These data show that glutamate release from megakaryocytes occurs in a SNARE-dependent, exocytotic manner and is increased during differentiation, suggesting that manipulation of glutamate signaling could influence megakaryocytopoiesis and, therefore, offer a suitable target for the treatment of thrombosis and other hematological disorders.