ArfGEF1/2 Inhibition Selectively Blocks Neurogenic ATP Release from Rat Tail Arteries

Introduction: Adenosine Triphosphate (ATP) is a versatile neurotransmitter that regulates vasomotor tone in the peripheral nervous systems. The Vesicular Nucleotide Transporter (VNUT) is thought to load ATP into vesicles that mediate purinergic neurotransmission and is unique amongst its relatives. Bioinformatics and AI-modelling in our lab shows that mammalian VNUT contains an endoplasmic reticulum (ER)-Golgi retention sequence and lacks synaptic vesicle and lysosome targeting motifs present in VNUT’s relatives. Our work in cultured cells suggests that VNUT localizes to sorting compartments akin to endosomes, ER and Golgi, as opposed to traditional acidic synaptic vesicles, like those that contain catecholamines. Critically, little is known of the molecular basis of differential release and recycling of purinergic and adrenergic vesicles in sympathetic neurons. We hypothesized that inhibiting ADP-ribosylation factor guanine nucleotide exchange factors (ArfGEFs) GBF1, ArfGEF1, and ArfGEF2 would preferentially attenuate purinergic signalling in contrast to adrenergic (norepinephrine) signalling in the rat tail artery, consistent with VNUT localization to an endosomal/Golgi/ER-resembling compartment. Methods: Male and female rat tail arteries were studied by field-stimulated wire myography and epifluorescence immunofluorescence microscopy. Results: Inhibiting ArfGEFs 1 and 2 selectively attenuated electrically-stimulated ATP release from the perivascular nerves of rat tail arteries while leaving adrenergic release unaffected. This effect was not use-dependent. Brefeldin-A, an inhibitor of ArfGEFs 1, 2, and GBF1, attenuated ATP-dependent arterial tension. Golgicide-A – inhibitor of GBF1 – did not significantly attenuate tension, indicating a likely role for ArfGEF1 and or ArfGEF2 in the maintenance of releasable ATP stores in rat tail artery. Immunostaining confirmed that VNUT localizes to perivascular nerves and does not colocalize with traditional vesicle markers (e.g. Syt1, SV2A). In addition, we observed markers of ER (calreticulin) and Golgi satellites in perivascular nerves, with partial colocalization of VNUT with trans-golgi markers. We also observed sex-differences in ATP-dependent vasomotor responses, where male rat arteries demonstrated greater ATP-driven vasomotor responses compared to female tissues. Conclusions: These findings demonstrate the first report of differential dependence of ATP and NE release on vesicle trafficking regulators and adaptor proteins. In addition, we show functional evidence supporting VNUT localization to an uncharacterized compartment that requires ArfGEFs 1 and/or 2 pathways to load ATP into vesicles. Observed sex differences in neurogenic vasomotor tone are consistent with recent reports studying mesenteric arteries. This work was supported by grant RGPIN-2019-04925 from the Natural Sciences and Engineering Research Council (NSERC) of Canada. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.