VEGF is a potent vasodilator, angiogenic and vascular permeability factor. VEGF has been shown to increase permeability through activation of calcium influx into the endothelial cell cytoplasm. Furthermore, transfection of a non-endothelial cell line with both VEGF-R2 and the non-specific cation channel TRP6 resulted in a significant increase in calcium upon addition of VEGF (Foster et al. 2002). To determine whether TRP6 was involved in the mechanism of VEGF-mediated calcium influx, we have measured the effects of a differential modulator of the TRP channels, flufenamic acid (FFA) on the VEGF-mediated increase in permeability. Although FFA has actions on other targets as well, it inhibits TRP3 and enhances the activation of TRP6.

Frogs ( Rana temporaria ) were anaesthetised by submersion in MS222 (1 mg ml^-1) and anaesthesia maintained by constant superfusion of the mesentery with frog Ringer solution containing MS222 (0.2 mg ml^-1). L_p (mean ± S.E.M. X 10^-7 cm s^-1 cmH₂O^-1) was measured during perfusion with 1 nM VEGF without or with 100 µM FFA in 20-30 µm diameter mesenteric microvessels, using a modification of the Landis Michel method (Michel et al. 1974). The vessel was then washed out for 20 min with 1 % BSA and L_p measured during VEGF perfusion with or without FFA, respectively. At the end of the experiment frogs were killed by cranial destruction. Figure 1 shows the effects of FFA and VEGF on two vessels. In Fig. 1A, VEGF alone resulted in a significant increase in L_p from 0.5 to 11.8. In the presence of 100 µM FFA, VEGF only increased L_p from 0.7 to 1.5, i.e. a significant inhibition. In Fig. 1B VEGF increased L_p only slightly, from 0.8 to 1.7. However, after 10 min perfusion with FFA, VEGF increased L_p from 1.9 to 10.0. This pattern was typical and overall, in eleven vessels, five gave a response to VEGF that was inhibited by FFA, and six a response that was enhanced by FFA. The mean baseline L_p (during perfusion with BSA or FFA) was significantly lower in vessels in which FFA had an inhibitory effect (1.2 ± 0.8) rather than an enhancing effect (3.4 ± 0.9). When the vessel groups were analysed separately, in vessels in which VEGF caused a weak increase in L_p (2.3 ± 0.4-fold), FFA perfusion caused a significant enhancement of the response (to 3.6 ± 0.6-fold, P < 0.01, paired t test). This was greater than the response in the presence of FFA in the inhibitory vessels (1.2 ± 0.3-fold, P < 0.02, t test). In summary these data show that VEGF can either increase or decrease L_p during FFA perfusion, supporting the hypothesis that different TRP channels regulate VEGF-mediated permeability.

This work was supported by the BHF (BB2000030 and PG98023).