Astrocytes Control Breathing Through pH-Dependent Vesicular Release of Atp

Abstract

Extracellular signalling mediated by ATP has been associated with central chemosensory function. It has been shown that increase in pCO2 in the arterial blood triggers an immediate release of ATP from the chemosensitive regions located on the ventral surface of the medulla oblongata (VMS).Using in vitro and in vivo preparations and novel genetically encoded Ca2+ indicator based on cyclically permutated GFP - Case 12, we show that astrocytes in the ventral regions of the medulla oblongata are highly sensitive to changes in pH. Decrease in extracellular pH from 7.4 to 7.2 induced transient increases in [Ca2+]i in astrocytes from dissociated neuro-glial cultures prepared from the VMS, in ventral astrocytes of organotypic brainstem slice cultures and in acute horizontal brainstem slices of adult rats, as well as on the VMS in anaesthetized and artificially ventilated rats. ATP receptor antagonists such as MRS2179, PPADS and TNP-ATP effectively blocked [Ca2+]i responses evoked by lowering pH in VMS astrocytes. ATP hydrolyzing enzyme apyrase completely prevented propagation of [Ca2+]i excitation in VMS astrocytes evoked by lowering external pH. These data suggest that Ca2+ responses induced by lowering external pH are mediated by ATP release and subsequent activation of P2 receptors. Inhibitors of vesicular transport brefeldine A and bafilomycin A both effectively abolished [Ca2+]i excitation of VMS astrocytes evoked by lowering external pH. Incubation of astrocytes with FM 1-46 dye (vesicular marker) leads to selective labelling of intracellular vesicles. Acidification of external medium induced decrease in the fluorescence intensity of vesicles labelled with FM 1-43. These data suggest that VMS astrocytes respond to a decrease in pH by releasing ATP via vesicular exocytosis.