Characterization of endothelin receptor subtypes mediating Ca 2+ mobilization and contractile response in rabbit iris dilator muscle

1 We investigated the characteristics of endothelin (ET)‐induced contraction and changes in intracellular Ca 2+ concentration ([Ca 2+ ] i ) using the fura‐2‐loaded and non‐loaded rabbit iris dilator. ET‐1 and ET‐2 (3–100 nM) and ET‐3 (30–1000 nM) caused contraction in a concentration‐dependent fashion. 2 The selective ET B ‐receptor agonists, IRL1620 and sarafotoxin S6c produced only a small contraction or no contraction at a concentration of 1 μ. The rank order of potencies for the contraction (pD 2 value) was ET‐1 = ET‐2 > ET‐3 » sarafotoxin S6c = IRL1620. 3 The contractile response to ET‐3 was antagonized by pretreatment with BQ‐123 (10 nM), a selective ET A receptor antagonist. The contractile responses to ET‐1 and ET‐2 were antagonized by pretreatment with BQ‐123 (10 μ), but not at a concentration of 10 nM. 4 ETs increased [Ca 2+ ] i and sustained muscle contraction. ET‐1 (100 nM), ET‐2 (100 nM), and ET‐3 (1 μ) induced an elevation of [Ca 2+ ] i consisting of two components: first a rapid and transient elevation to reach a peak, followed by a second, sustained elevation; a sustained contraction was produced without a transient contraction. The ET B receptor‐selective agonist, IRL1620 (1 μ) and sarafotoxin S6c (1 μ) also induced a rapid and transient elevation of [Ca 2+ ] i to reach a peak and a sustained elevation, together with only a small contraction or no contraction. 5 ET‐1 (100 nM) induced a transient increase in [Ca 2+ ] i in a Ca 2+ ‐free, 2 m M EGTA‐containing physiological saline solution (Ca 2+ ‐free PSS), and a small sustained contraction which was significantly different from that induced by ET‐1 (100 nM) in normal PSS. The ET‐1‐induced increase in [Ca 2+ ] i and sustained contraction were not affected by the voltage‐dependent Ca 2+ channel blocker, nicardipine (10 μ). The ET‐1‐induced transient increase in [Ca 2+ ] i was significantly reduced by the sarcoplasmic reticulum (SR) Ca 2+ ‐ATPase inhibitor, cyclopiazonic acid (30 μ); however, the ET‐1‐induced sustained contraction was not affected by this agent. 6 The selective ET A receptor antagonist, BQ‐123 (100 nM) reduced the ET‐3 (100 nM)‐induced contraction, but did not affect the transient increase or elevation of the second phase of [Ca 2+ ] i . However, this antagonist at 1 μ did not affect the ET‐1 (100 nM)‐ and ET‐2 (100 nM)‐induced elevation of [Ca 2+ ] i and contractile response, or the IRL1620‐induced elevation of [Ca 2+ ] i . 7 The selective ET B receptor antagonist, BQ‐788 (1 μ) reduced the transient increase in [Ca 2+ ] i induced by ET‐1 (30 nM), ET‐2 (30 nM), ET‐3 (100 nM) and IRL1620 (1 μ), but did not affect the sustained elevation of [Ca 2+ ] i and contractile responses produced by ET‐1, ET‐2 and ET‐3. 8 Pretreatment with IRL1620 (1 μ) reduced the increase in [Ca 2+ ] i induced by IRL1620 (1 μ) and sarafotoxin S6c (1 μ), as well as the ET‐1 (100 nM)‐, ET‐2 (100 nM)‐ and ET‐3 (1 μ)‐induced elevation of [Ca 2+ ] i , whereas in the presence of IRL1620, ET‐1‐, ET‐2‐ and ET‐3‐induced contractions were unaltered. 9 These results suggest that ET A and ET B receptor subtypes exist in the rabbit iris dilator muscle, and that the ET A receptor is divided into: (1) BQ‐123‐sensitive ET A subtypes activated by ET‐1, ET‐2 and ET‐3, and (2) BQ‐123‐insensitive ET A subtypes activated by ET‐1 and ET‐2, which cause the sustained increase of [Ca 2+ ] i and contraction; in contrast, ET B receptor subtypes, are activated by ET‐1, ET‐2, ET‐3, IRL1620 and sarafotoxin S6c and cause the transient and sustained increase in [Ca 2+ ] i which is not able to contract the smooth muscle.