J Physiol 587.22 PIP3 and PIP2 activation of vascular TRPC1 5367 stimulation but is unlikely to be involved in activating native TRPC1 channels through stimulation of ETA receptors. Therefore we investigated the effects of several established inhibitors of other phospholipases that may be involved in ETA receptor-mediated activation of TRPC1 channel currents. Figure 3C shows that pharmacological inhibitors of phosphatidylcholine-PLC (PC-PLC, 100 μM D-609, n = 5), cytosolic and Ca2+-dependent and -independent forms of phospholipase A2 (PLA2, 100 μM AACOCF3, n = 4 and 100 μM PACOCF3, n = 4) and phospholipase D (PLD, 100 μM C2-ceramide, n = 6) had no effect on ETA receptor-mediated TRPC1 channel activity. Stimulation of ETA receptors has also been shown to activate Rho kinase (Ivey et al. 2008) but Fig. 3C shows that the Rho kinase inhibitors HA-110 (5 μM, n = 5) and Y27632 (1 μM, n = 5) had no effect on ETA receptor-mediated TRPC1 channel activity. These data suggest that PC-PLC, PLA2, PLD and Rho kinase are also not involved in TRPC1 channel activation initiated by ETA receptor stimulation. Previous studies have shown that stimulation of ETA receptors can activate phosphoinositol-3-kinase (PI-3-kinase), which phosphorylates PIP2 to form PIP3, with the latter phospholipid capable of stimulating PKC activity (see review by Ivey et al. 2008). Therefore we investigated the role of a PI-3-kinase-mediated mechanism on ETA and ETB receptor-mediated TRPC1 channel activity in cell-attached patches using selective concentrations of wortmannin and a structurally different compound, PI-828, which both inhibit PI-3-kinase. Figure 4A shows that 50 nM wortmannin signiﬁcantly reduced mean NPo of ETA receptor-mediated TRPC1 channel activity from 0.23 ± 0.06 to 0.02 ± 0.01 (94 ± 4% inhibition, n = 6, P < 0.01). In addition Fig. 4B shows that 3 μM PI-828 also signiﬁcantly attenuated mean NPo of ETA receptor-mediated TRPC1 channel activity from 0.14 ± 0.04 to 0.02 ± 0.01 (85 ± 5% inhibition, n = 5, P < 0.01). Importantly, Fig. 4C and D illustrates that 50 nM wortmannin (control mean NPo of 0.18 ± 0.06 and 0.18 ± 0.07 in wortmannin, n = 6) and 3 μM PI-828 (control mean NPo of 0.31 ± 0.11 and 0.24 ± 0.08 in PI-828, n = 6) had no effect on ETB receptor-mediated TRPC1 channel activity indicating that these reagents do not have direct non-speciﬁc effects on TRPC1 channel currents. These results provide evidence that stimulation of ETA and ETB receptors evokes TRPC1 channel activity via different signal transduction mechanisms. Stimulation of ETA receptors is coupled to TRPC1 channels via a PI-3-kinase-dependent pathway whereas a PI-PLC-dependent pathway links ETB receptor-mediated TRPC1 channel opening, and both these pathways are likely to induce TRPC1 channel activity through a PKC-dependent mechanism. Figure 4. Stimulation of ETA receptors activates TRPC1 channel currents via a PI-3-kinase-mediated pathway in cell-attached patches A and B show that ETA receptor-mediated TRPC1 channel activity was inhibited by co-application of 50 nM wortmannin or 3 μM PI-828, whereas C and D show that these compounds had no effect on ETB receptor-mediated TRPC1 channel activity. C 2009 The Authors. Journal compilation C 2009 The Physiological Society