5366 S. N. Saleh and others J Physiol 587.22 NPo of ETB receptor-mediated channel activity, activated by 100 nM ET-1 in the presence of 100 nM BQ-123, was also signiﬁcantly inhibited from 0.31 ± 0.08 to 0.05 ± 0.02 (84 ± 3% inhibition, n = 7, P < 0.01) by 3 μM chelerythrine. Figure 2C illustrates that ETA receptor-mediated channel activity in cell-attached patches was maintained following excision into the inside-out conﬁguration and that bath application of anti-TRPC1 antibodies to the cytosolic surface of these inside-out patches signiﬁcantly reduced mean NPo of ETA receptor-mediated activity from 0.19 ± 0.03 to 0.01 ± 0.01 (95 ± 5% inhibition, n = 5, P < 0.01). Moreover Fig. 2D shows that anti-TRPC1 anti- bodies also signiﬁcantly inhibited the mean NPo of ETB receptor-mediated channel activity from 0.22 ± 0.08 to 0.03 ± 0.02 (87 ± 8% inhibition, n = 5, P < 0.01). In control experiments, Fig. 2E and F show that following pre-incubation with their antigenic peptide, anti-TRPC1 antibodies had no effect on ETA receptor-mediated or ETB receptor-mediated channel activity (n = 4 for each). Channel activity often recovered, at least partially, following washout of anti-TRPC1 antibodies indicating some degree of reversibility in the conditions used. These data show that stimulation of both ETA and ETB receptors activates TRPC1 channel currents through a PKC-dependent mechanism in coronary artery myocytes. Distinct signalling pathways mediate ETA and ETB receptor stimulation of TRPC1 channel activity In the next series of experiments we investigated the signalling pathways linking ETA and ETB receptors to PKC-mediated opening of TRPC1 channels. ETA and ETB G-protein-coupled receptors can be linked to different phospholipases that generate the endogenous PKC activator diacylglycerol (DAG, Ivey et al. 2008). Therefore we investigated the effect of biochemically characterised pharmacological inhibitors of endogenous phospholipases on ETA and ETB receptor-mediated TRPC1 channel activity in cell-attached patches. Figure 3A shows that the phosphoinositol- phospholipase C (PI-PLC) inhibitor U73122 (2 μM) signiﬁcantly inhibited the mean NPo of ETB receptor- mediated TRPC1 channel activity from 0.26 ± 0.05 to 0.04 ± 0.02 (89 ± 4% inhibition, n = 7, P < 0.01) whereas Fig. 3B demonstrates that this PI-PLC inhibitor had no effect on ETA receptor-mediated TRPC1 channel activity (control mean NPo was 0.21 ± 0.06 and 0.18 ± 0.05 in U73122, n = 7). In addition, 2 μM U73343, an inactive analogue of U73122, had no effect on ETB receptor-mediated TRPC1 channel activity (n = 4, data not shown). The above studies indicate that a PI-PLC-mediated mechanism couples ETB receptors to TRPC1 channel Figure 3. Stimulation of ETB, but not ETA, receptors activates TRPC1 channel currents via a PLC-mediated transduction pathway in cell-attached patches A, ETB receptor-mediated TRPC1 channel activity was inhibited by co-application of 2 μM U73122 at −70 mV. B, ETA receptor-mediated TRPC1 channel activity was unaffected by co-application of 2 μM U73122. C, mean data showing that ETA receptor-mediated TRPC1 channel activity (ET-1-evoked NPo in the presence of 100 nM BQ-788) at −70 mV was also unaffected by co-application of inhibitors against different phospholipases and Rho kinase (see text for details). C 2009 The Authors. Journal compilation C 2009 The Physiological Society