Vascular endothelial growth factor-A (VEGF) is spliced into two different isoform families, VEGF-Axxxa and VEGF-Axxxb, differing in only the C-terminus. VEGF-A165a promotes and VEGF-A165b inhibits angiogenesis1. VEGF receptors trigger downstream changes in Ca2+ flux via TRP channels in endothelial cells2. New evidence shows that the pronociceptive actions of VEGF-A165a are mediated by TRPV13. This has implications for proposed VEGF treatments for diabetic neuropathy. Our aim was to investigate the effect of VEGF isoforms on intracellular Ca2+ in sensory neurons. Single cell Ca2+ imaging. Rat dorsal root ganglia (DRG) were isolated and plated on glass cover slips, and loaded with Fura-2-AM the following day. Human haemangioma endothelial cells (HemECs) > passage 4 were cultured on glass cover slips, and loaded as above (positive control for VEGF activity). Cells were perfused with 3nM VEGF165a or b, followed by positive control (100 µM ATP/HemEC, 100nM capsaicin/DRG). Cells were maintained at 35°C. Relative Ca2+ concentrations were measured by the ratio of 340/380nm fluorescence. High throughput Ca2+ assay. DRG were cultured on black-sided 96 well plates, and pre-incubated with 1µM SPHINX-31 or 2.5nM VEGF-A165b. 24 hours later, DRG were loaded with Fluo-4, and stimulated with a range of capsaicin concentrations at 37 ° C. Fluo-4 was excited at 494 nm and emissions at 516 nm were recorded. Data are presented relative to vehicle control ± SEM. 2.5nM VEGF-A165a or VEGF-A165b evoked increases in calcium concentration over vehicle in single HemECs (38.04±1.05-fold, and 11.58±1.00-fold respectively n=100-200). 100µM ATP caused an increase of 12.82±0.85-fold (n=200, all p<0.0001, 1-way ANOVA). Neither VEGF-A165a nor VEGF-A165b acutely changed intracellular Ca2+ in single primary DRG cells (0.22±0.09, and 0.79±0.54, n=57-120), nor caused any difference in Ca2+ concentration increases triggered by subsequent 100nM capsaicin after 5 min (5.56±2.01-fold vs. 7.81±1.14-fold, n=9, 25). With the high-throughput assay, 24hr incubation of DRGs with VEGF-A165b reduced the concentration response to capsaicin (n=5-6). Incubation with the SPRK1 inhibitor SPHINX-31 (1µM), which favours splicing to VEGF-A165b, also decreased responses to 1µM capsaicin under high glucose (65mM) conditions (capsaicin alone AUC= 5.85±0.95s, 1µM capsaicin + 1µM SPHINX-31 AUC= 1.83±0.59s, n=4, p<0.05, 2-way ANOVA). VEGF isoforms do not directly trigger Ca2+ release in primary DRG neurons[NB5] . However, overnight stimulation with VEGF-A165b can reduce DRG Ca2+ currents elicited by capsaicin via TRPV1 receptors, and this effect can be mimicked in high glucose conditions by the splice factor kinase inhibitor SPHINX-31. These results support the hypothesis that splice specific VEGF isoform control could provide a mechanism for inhibition of sensory sensitisation.