Activation of native TRPC1/C5/C6 channels by endothelin-1 is mediated by both PIP3 and PIP2 in rabbit coronary artery myocytes

We investigate activation mechanisms of native TRPC1/C5/C6 channels (termed TRPC1 channels) by stimulation of endothelin-1 (ET-1) receptor subtypes in freshly dispersed rabbit coronary artery myocytes using single channel recording and immunoprecipitation techniques. ET-1 evoked non-selective cation channel currents with a unitary conductance of 2.6 pS which were not inhibited by either ET(A) or ET(B) receptor antagonists, respectively BQ-123 and BQ788, when administered separately. However, in the presence of both antagonists, ET-1-evoked channel activity was abolished indicating that both ET(A) and ET(B) receptor stimulation activate this conductance. Stimulation of both ET(A) and ET(B) receptors evoked channel activity which was inhibited by the protein kinase C (PKC) inhibitor chelerythrine and by anti-TRPC1 antibodies indicating that activation of both receptor subtypes causes TRPC1 channel activation by a PKC-dependent mechanism. ET(A) receptor-mediated TRPC1 channel activity was selectively inhibited by phosphoinositol-3-kinase (PI-3-kinase) inhibitors wortmannin (50 nm) and PI-828 and by antibodies raised against phosphoinositol-3,4,5-trisphosphate (PIP(3)), the product of PI-3-kinase-mediated phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP(2)). Moreover, exogenous application of diC8-PIP(3) stimulated PKC-dependent TRPC1 channel activity. These results indicate that stimulation of ET(A) receptors evokes PKC-dependent TRPC1 channel activity through activation of PI-3-kinase and generation of PIP(3). In contrast, ET(B) receptor-mediated TRPC1 channel activity was inhibited by the PI-phospholipase C (PI-PLC) inhibitor U73122. 1-Oleoyl-2-acetyl-sn-glycerol (OAG), an analogue of diacylglycerol (DAG), which is a product of PI-PLC, also activated PKC-dependent TRPC1 channel activity. OAG-induced TRPC1 channel activity was inhibited by anti-phosphoinositol-4,5-bisphosphate (PIP(2)) antibodies and high concentrations of wortmannin (20 μm) which depleted tissue PIP(2) levels. In addition exogenous application of diC8-PIP(2) activated PKC-dependent TRPC1 channel activity. These data indicate that stimulation of ET(B) receptors evokes PKC-dependent TRPC1 activity through PI-PLC-mediated generation of DAG and requires a permissive role of PIP(2). In conclusion, we provide the first evidence that stimulation of ET(A) and ET(B) receptors activate native PKC-dependent TRPC1 channels through two distinct phospholipids pathways involving a novel action of PIP(3), in addition to PIP(2), in rabbit coronary artery myocytes