Pharmacological profile of phosphatidylinositol 3-kinases and related phosphatidylinositols mediating endothelin_A receptor-operated native TRPC channels in rabbit coronary artery myocytes

BACKGROUND AND PURPOSE Endothelin_A (ET_A) receptor-operated canonical transient receptor potential (TRPC) channels mediate Ca²⁺ influx pathways, which are important in coronary artery function. Biochemical pathways linking ET_A receptor stimulation to TRPC channel opening are unknown. We investigated the involvement of phosphatidylinositol 3-kinases (PI3K) in ET_A receptor activation of native heteromeric TRPC1/C5/C6 and TRPC3/C7 channels in rabbit coronary artery vascular smooth muscle cells (VSMCs). EXPERIMENTAL APPROACH A pharmacological profile of PI3K was created by studying the effect of pan-PI3K, pan-Class I PI3K and Class I PI3K isoform-selective inhibitors on ET_A receptor-evoked single TRPC1/C5/C6 and TRPC3/C7 channel activities in cell-attached patches from rabbit freshly isolated coronary artery VSMCs. The action of phosphatidylinositol 3-phosphate- [PI(3)P], 4-phosphate- [PI(4)P] and 5-phosphate- [PI(5)P] containing molecules involved in PI3K-mediated reactions were studied in inside-out patches. Expression of PI3K family members in coronary artery tissue lysates were analysed using quantitative PCR. KEY RESULTS ET_A receptor-operated TRPC1/C5/C6 and TRPC3/C7 channel activities were inhibited by wortmannin. However, ZSTK474 and AS252424 reduced ET _A receptor-evoked TRPC1/C5/C6 channel activity but potentiated TRPC3/C7 channel activity. All the PI(3)P-, PI(4)P- and PI(5)P-containing molecules tested induced TRPC1/C5/C6 channel activation, whereas only PI(3)P stimulated TRPC3/C7 channels. CONCLUSIONS AND IMPLICATIONS ET_A receptor-operated native TRPC1/C5/C6 and TRPC3/C7 channel activities are likely to be mediated by Class I PI3KΥ and Class II/III PI3K isoforms, respectively. ET_A receptor-evoked and constitutively active PI3KΥ-mediated pathways inhibit TRPC3/C7 channel activation. PI3K-mediated pathways are novel regulators of native TRPC channels in VSMCs, and these signalling cascades are potential pharmacological targets for coronary artery disease.