Involvement of Ca2+ Channels in Endothelin–1 (ET–1)–Induced MAP Kinase Phosphorylation, Myosin Light Chain (MLC) Phosphorylation and Contraction in Rabbit Iris Sphincter

Abstract

Abstract: : Purpose: The goal of this study was to investigate the role and type of Ca²⁺ channels involved in the stimulatory effects of ET–1 on the Ca²⁺–dependent functional responses, p42/p44 MAP kinase phosphorylation, 20–kDa MLC phosphorylation and contraction, in rabbit iris sphincter. Methods: Myo–[³H]inositol phosphates production was measured by ion–exchange chromatography, MAP kinase phosphorylation was determined by Western blotting, MLC kinase (MLCK) activity was measured by incorporation of ³²Pi into MLC, and changes in muscle tension were recorded isometrically. Results: ET–1 induced inositol phosphates production, MAP kinase phosphorylation, MLC phosphorylation and contraction in a concentration–dependent manner with EC₅₀ values of 71, 8, 6 and 25 nM, respectively. ET–1–induced MAP kinase phosphorylation, MLC phosphorylation and contraction were not significantly affected by nifedipine (1–60 µM), an L–type Ca²⁺ channel blocker, or by LOE 908 (1–100 µM), a blocker of Ca²⁺–permeable nonselective cation channels. However, SKF96365, a receptor–operated Ca²⁺ channel (ROCC) blocker, inhibited MAP kinase phosphorylation, MLC phosphorylation and contraction in a concentration–dependent manner with IC₅₀ values of 28, 30 and 42 µM, respectively. 2–APB, a store–operated Ca²⁺ channel (SOCC) blocker, inhibited ET–1–induced MLC phosphorylation and contraction in a concentration–dependent manner with IC₅₀ values of 12.7 and 19 µM, respectively, but was without effect on MAP kinase phosphorylation. The combined effects of submaximal concentrations of SKF96365 and 2–APB on ET–1–induced MLC phosphorylation and contraction were not additive, implying that their inhibitory actions could be mediated through a common Ca²⁺ entry channel. Conclusion: The present study demonstrated for the first time that in rabbit iris sphincter (a) ET–1, through the ET_A receptor, stimulates MAP kinase phosphorylation, MLC phosphorylation and contraction in a concentration–dependent manner, (b) that these Ca²⁺–dependent functional responses are not significantly affected by nifedipine or LOE908, and (c) that ET–1– induced MLC phosphorylation and contraction are inhibited by SKF96365 and 2–APB, suggesting that these effects are mainly due to store–and/or receptor Ca²⁺ entry.

Keywords: second messengers: pharmacology/physiology • phosphorylation • calcium