Search

The present work investigated interactions between TRPC1/C5 and TRPC6 cation channel activities evoked by angiotensin II (Ang II) in native rabbit mesenteric artery vascular smooth muscle cells (VSMCs). In low intracellular Ca2+ buffering conditions (0.1 m m BAPTA), 1 n m and 10 n m Ang II activated both 2 pS TRPC1/C5 channels and 15-45 pS TRPC6 channels in the same outside-out patches. However, increasing Ang II to 100 n m abolished TRPC6 activity but further increased TRPC1/C5 channel activity. Comparison of individual patches revealed an inverse relationship between TRPC1/C5 and TRPC6 channel activity suggesting that TRPC1/C5 inhibits TRPC6 channel activity. Inclusion of anti-TRPC1 and anti-TRPC5 antibodies, raised against intracellular epitopes, in the patch pipette solution blocked TRPC1/C5 channel currents but potentiated by about six-fold TRPC6 channel activity evoked by 1-100 n m Ang II in outside-out patches. Bath application of T1E3, an anti-TRPC1 antibody raised against an extracellular epitope, also increased Ang II-evoked TRPC6 channel activity. With high intracellular Ca2+ buffering conditions (10 m m BAPTA), 10 n m Ang II-induced TRPC6 channel activity was increased by about five-fold compared to channel activity with low Ca2+ buffering. In addition, increasing intracellular Ca2+ levels ([Ca2+]i) at the cytosolic surface inhibited 10 n m Ang II-evoked TRPC6 channel activity in inside-out patches. Moreover, in zero external Ca2+ (0 [Ca2+]o) 100 n m Ang II induced TRPC6 channel activity in outside-out patches. Pre-treatment with the PKC inhibitor, chelerythrine, markedly increased TRPC6 channel activity evoked by 1-100 n m Ang II and blocked the inhibitory action of [Ca2+]i on TRPC6 channel activity. Co-immunoprecipitation studies shows that Ang II increased phosphorylation of TRPC6 proteins which was inhibited by chelerythrine, 0 [Ca2+]o and the anti-TRPC1 antibody T1E3. These results show that TRPC6 channels evoked by Ang II are inhibited by TRPC1/C5-mediated Ca2+ influx and stimulation of PKC, which phosphorylates TRPC6 subunits. These conclusions represent a novel interaction between two distinct vasoconstrictor-activated TRPC channels expressed in the same native VSMCs. [ABSTRACT FROM AUTHOR]

In the present work we used patch pipette techniques to study the properties of a novel Ca2+-permeable cation channel activated by the potent coronary vasoconstrictor endothelin-1 (ET-1) in freshly dispersed rabbit coronary artery myocytes. With cell-attached recording bath application of 10 nm ET-1 evoked cation channel currents ( Icat) with subconductance states of about 18, 34 and 51 and 68 pS, and a reversal potential of 0 mV. ET-1 evoked channel activity when extracellular Ca2+ was the charge carrier, illustrating significant Ca2+ permeability. ET-1-induced responses were inhibited by the ETA receptor antagonist BQ123 and the phospholipase C (PLC) inhibitor U73122. The diacylglycerol analogue 1-oleoyl-2-acetyl- sn-glycerol (OAG) also stimulated Icat, but the protein kinase C (PKC) inhibitor chelerythrine did not inhibit either the OAG- or ET-1-induced Icat. Inositol 1,4,5-trisphosphate (IP3) did not activate Icat, but greatly potentiated the response to OAG and this effect was blocked by heparin. Bath application of anti-TRPC3 and anti-TRPC7 antibodies to inside-out patches markedly inhibited ET-1-evoked Icat, but antibodies to TRPC1, C4, C5 and C6 had no effect. Immunocytochemical studies demonstrated preferential TRPC7 expression in the plasmalemma, whereas TRPC3 was distributed throughout the myocyte, and moreover co-localization of TRPC3 and TRPC7 signals was observed at, or close to, the plasma membrane. Flufenamic acid, Gd3+, La3+ and extracellular Ca2+ inhibited Icat with IC50 values of 2.45 μm, 3.8 μm, 7.36 μm and 22 μm, respectively. These results suggest that in rabbit coronary artery myocytes ET-1 evokes a Ca2+-permeable non-selective cation channel with properties similar to TRPC3 and TRPC7, and indicates that these proteins may be important components of this conductance. [ABSTRACT FROM AUTHOR]