Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC36

Poster Communications

Vesicular release of ATP in brainstem astrocytes evoked by pH changes

V. Kasymov1, C. Castaldo3, N. Marina1, S. Kasparov2, A. Gourine1

1. Neuroscience, Physiology & Pharmacology, University College London, London, United Kingdom. 2. Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom. 3. Universit

The mechanisms of central nervous chemosensory function underlying detection of blood and brain pH levels are not completely understood. We have recently demonstrated that a small decrease in extracellular pH from 7.4 to 7.2 induces rapid increase in [Ca2+]i in astrocytes located in the central chemosensitive area located at the ventral medullary surface (VMS) (Gourine et al. 2010). These increases were largely mediated by release of ATP. Here we sought to identify the mechanism of ATP release triggered by acidification. All experiments were performed on Sprague-Dawley rats (UCL breeding colony, London, UK). Methods for tissue culturing and preparation of slices have been described in (Gourine 2010). All recordings were performed at 35-37oC in HBSS. Acidification-induced [Ca2+]i responses in VMS astrocytes were not affected by either carbenoxolone (10 µM, blocker pannexin hemichannels) or lanthanum (La3+; 100 µM - a connexin hemichannel blocker that does not affect gap junctions). However, both brefeldine A and bafilomycin A - inhibitors of vesicular transport and vesicular H+ ATPase, effectively abolished [Ca2+]i excitation of VMS astrocytes evoked by lowering external pH. Given that these Ca2+ responses are mediated by ATP (Gourine et al 2010), these observations indicate that acidification leads to release of ATP via vesicular expcytosis. We further investigated the mechanisms of pH-induced vesicular events in VMS astrocytesusing confocal and total internal reflection (TIRF) microscopy. Astrocytes were identified by expression of green or red fluorescent protein Case12 and DsRed respectively (Gourine et al 2010). Incubation of VMS astrocytes in primary culture with fluorescent vesicular markers FM 1-43 and Quinacrine (Kreda et al 2007), or Mant-ATP (a fluorescent nucleotide analogue) labelled populations of vesicles in astrocytes in cultures from the central chemosensitive area. Acidification of external medium induced rapid destaining of FM 1-43, Quinacrine or Mant-ATP loaded vesicles. To test whether this occurs in pH stimulated VMS astrocytes, dissociated VMS neuro-glial cultures were incubated with a UV-excitable dye Cascade blue conjugated with dextran (3000 MW). This complex is too large to enter the cell directly and should only become internalised via “kiss and run” exocytosis (Chen 2005). Decrease in extracellular pH from 7.4 to 7.0 induced the labelling of vesicles in VMS astrocytes. These data demonstrate a consistent with the notion of vesicular release of ATP triggered by acidification. ATP released in this manner then propagates Ca2+ excitation across the astrocytic network to provide excitatory tone to the respiratory rhythm generator.

Where applicable, experiments conform with Society ethical requirements