VNUT and VMAT2 segregate within sympathetic varicosities and localize near preferred CaV2 isoforms in rat tail artery
American Journal of Physiology. Heart and Circulatory Physiology
ATP and norepinephrine (NE) are co-released from peripheral sympathetic nerve terminals. Whether they are stored in the same vesicles has been debated for decades. Preferential dependence of NE or ATP release on Ca2+ influx through specific CaV2 isoforms suggests that NE and ATP are stored in separate vesicle pools, but simultaneous imaging of NE and ATP containing vesicles within single varicosities has not been reported. We conducted immunohistochemical studies of Vesicular Monoamine
... er 2 (VMAT2/SLC18A2) and Vesicular Nucleotide Translocase (VNUT/SLC17A9) as markers of vesicles containing NE and ATP in sympathetic nerves of the rat tail artery. A large fraction of varicosities exhibited neighbouring, rather than overlapping, VNUT and VMAT2 fluorescent puncta. VMAT2 but not VNUT colocalized with synaptotagmin 1 (Syt1). CaV2.1, CaV2.2 and CaV2.3 are expressed in nerves in the tunica adventitia. VMAT2 preferentially localized adjacent to CaV2.2 and CaV2.3, rather than CaV2.1. VNUT preferentially localized adjacent to CaV2.3 > CaV2.2 >> CaV2.1. Using wire myography, inhibition of field-stimulated vasoconstriction with the CaV2.3 blocker SNX-482 (0.25 µM) mimicked the effects of the P2X inhibitor suramin (100 µM) rather than the α-adrenergic inhibitor phentolamine (10 µM). Variable sensitivity to SNX-482 and suramin between animals closely correlated with CaV2.3 staining. We conclude that a majority of ATP and NE stores localize to separate vesicle pools that employ different synaptotagmin isoforms and that localize near different CaV2 isoforms to mediate vesicle release. CaV2.3 appears to play a previously unrecognized role in mediating ATP release in the rat tail artery.