Adenosine triphosphate (ATP) is actively transported into vesicles for purinergic neurotransmission by the vesicular nucleotide transporter, VNUT, encoded by the gene, solute carrier 17, member 9 (SLC17A9). In this chapter, methods are described for fluorescent labeling of VNUT positive cells and quantification of vesicular ATP release using live cell imaging. Directions for preparation of viable dissociated neurons and cellular labeling with an antibody against VNUT and for ATP containing synaptic vesicles with fluorescent ATP markers, quinacrine or MANT-ATP, are detailed. Using confocal microscope live cell imaging, cells positive for VNUT can be observed colocalized with fluorescent ATP vesicular markers, which occur as discrete puncta near the cell membrane. Vesicular release, stimulated with a depolarizing, high potassium physiological saline solution induces ATP marker fluorescence reduction at the cell membrane and this can be quantified over time to assess ATP release. Pretreatment with the voltage gated calcium channel blocker, cadmium, blocks depolarization-induced membrane fluorescence changes, suggesting that VNUT-positive neurons release ATP via calcium-dependent exocytosis. This technique may be applied for quantifying vesicular ATP release across the peripheral and central nervous system and is useful for unveiling the intricacies of purinergic neurotransmission.
Burnstock G, Satchell DG, Smythe A (1972) A comparison of the excitatory and inhibitory effects of non-adrenergic, non-cholinergic nerve stimulation and exogenously applied ATP on a variety of smooth muscle preparations from different vertebrate species. Br J Pharmacol 46(2):234–242CrossRefGoogle Scholar
Ziogas J, Vessey K (2001) Angiotensin-induced enhancement of excitatory junction potentials evoked by periarteriolar nerve stimulation and vasoconstriction in rat mesenteric arteries are both mediated by the angiotensin AT1 receptor. Pharmacology 63(2):103–111. https://doi.org/10.1159/000056120CrossRefPubMedGoogle Scholar
Bodin P, Burnstock G (2001) Evidence that release of adenosine triphosphate from endothelial cells during increased shear stress is vesicular. J Cardiovasc Pharmacol 38(6):900–908CrossRefGoogle Scholar