Vesicular release of ATP by brainstem astrocytes evoked by changes in pH

Abstract

The mechanisms of central chemosensory function are not completely understood. Here we explored the mechanisms underlying release of ATP by brainstem astrocytes in response to changes in pH.RESULTSLoading of astrocytes with fluorescent ATP analogue MANT‐ATP resulted in abundant punctate fluorescent staining in the cytosol and this was prevented by bafilomycin A1, indicating vesicular accumulation of the marker. In brainstem astrocytes, TIRF imaging revealed progressive decreases in MANT‐ATP fluorescence intensity in response to a decrease in external pH indicating fusion of the labelled compartments. The number of fusion events triggered by acidification in brainstem astrocytes loaded with BAPTA‐AM was significantly reduced indicating that vesicular fusion requires intracellular Ca2+ signalling. The fusion rate in cortical astrocytes was not affected by acidification. Acidification‐evoked vesicular fusion responses in brainstem astrocytes were unaffected by blockade of ATP‐mediated signalling with MRS2179 or apyrase. Quantitative RT‐PCR analysis of genes encoding proteins associated with vesicular fusion revealed that expression levels of VAMP3, Slc1a2 and Slc17a9 are significantly higher in brainstem astrocytes as compared to cortical astrocytes.CONCLUSIONSChemosensory stimulation of brainstem astrocytes triggers vesicular fusion underlying release of ATP.