TDK conference 2019

Neuroinflammation has a key role in the pathogenesis of several diseases. This pathological process is mainly characterised by the activation of microglia as well as astrocytes producing different cytokines. Glutaminase is indirectly involved in these events, thus the inhibition of this enzyme may have therapeutical indication. Recently it has been shown that one of the phosphodiesterase (PDE) inhibitors inhibits glutaminase as well, however similar effect regarding to other PDE inhibitors is still unknown.

We created an in vitro model which is suitable for examining the inhibition of glutaminase in astrocytes. In our experiments we prepared primary rat astrocyte cultures and after becoming confluent, we selectively removed microglia with a chemical treatment in order to get highly purified astrocyte cultures. The effect of ammonia, zaprinast, theophylline and 6-diazo-5-oxo-L-norleucin (DON) on citotoxicity was assessed by measuring propidium iodide (PI) and neutral red uptake as well as lactate dehydrogenase activity. After this, the cells were incubated with or without ammonia and were exposed to zaprinast, theophylline and DON. The glutaminase activity measurement of these agents was carried out by glutamate assay kit based on colorimetric method.

The result of the cytotoxicity examination by PI staining was not reliable, however both the neutral red uptake and the LDH activity measurement were suitable for assessing the optimal concentration/time of the treatments regarding to the examined chemicals. The concentrations of 300 uM and 500 uM theophylline, 100 uM and 300 uM zaprinast, 100 uM DON were chosen to evaluate the action of these chemicals on the glutaminase inhibition. The changes of intracellular glutamate level after the treatments could be measured with spectrophotometry, however these preliminary findings require further investigations.