Pharmacological inhibition of V-ATPase reduces extracellular ATP and glutamate and delays neural tube closure in Xenopus laevis

Neurulation requires coordinated morphogenetic movements and extracellular signals for timely neural tube closure. Although purinergic and glutamatergic signaling has been implicated in this process, whether V-ATPase-dependent vesicular processes contribute to extracellular ATP and glutamate accumulation remains unclear. In Xenopus laevis embryos, we assessed the expression of vesicular-machinery components during neurulation and the effects of pharmacological V-ATPase inhibition with bafilomycin A1 (BafA1). Transcripts of VNUT, VGLUT1–3, V-ATPase subunit c, and SNARE-associated components were detected between stages 12.5 and 20 and VNUT and VGLUT1/2 immunoreactivity was also detected by Western blot. BafA1 exposure increased incomplete neural tube closure at stage 20 in a concentration-dependent manner, consistent with delayed closure, and reduced cumulative path length and mean speed of tracked pigmented cells in the dorsal neuroepithelial region. At stage 18, BafA1 reduced ATP and glutamate accumulation in whole-embryo-conditioned medium without detectable changes in whole-embryo intracellular content. NMDA co-treatment partially rescued incomplete closure at 0.125 and 0.25 µM BafA1 and neural tube opening across 0.125–0.5 µM BafA1, whereas ATP supplementation did not achieve the same effect. These findings are consistent with a contribution of V-ATPase-dependent processes to extracellular ATP and glutamate accumulation and timely neural tube closure, but vesicular loading and release were not measured directly.