Inhibition of ryanodine receptors by 4-(2-aminopropyl)-3,5-dichloro-N,N- dimethylaniline (FLA 365) in canine pulmonary arterial smooth muscle cells

Abstract

Ryanodine is a selective ryanodine receptor (RyR) blocker, with binding dependent on RyR opening. In whole-cell studies, ryanodine binding can lock the RyR in an open-conductance state, short-circuiting the sarcoplasmic reticulum, which restricts studies of inositol-1,4,5-trisphosphate receptor (InsP 3R) activity. Other RyR blockers have nonselective effects that also limit their utility. 4-(2-Aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365) blocks RyR-elicited Ca2+ increases in skeletal and cardiac muscle; yet, its actions on smooth muscle are unknown. Canine pulmonary arterial smooth muscle cells (PASMCs) express both RyRs and InsP3Rs; thus, we tested the ability of FLA 365 to block RyR- and serotonin-mediated InsP 3R-elicited Ca2+ release by imaging fura-2-loaded PASMCs. Acute exposure to 10 mM caffeine, a selective RyR activator, induced Ca 2+ increases that were reversibly reduced by FLA 365, with an estimated IC50 of ∼1 to 1.5 μM, and inhibited by 10 μM ryanodine or 10 μM cyclopiazonic acid. FLA 365 also blocked L-type Ca 2+ channel activity, with 10 μM reducing Ba2+ current amplitude in patch voltage-clamp studies to 54 ± 6% of control and 100 μM FLA 365 reducing membrane current to 21 ± 6%. InsP 3R-mediated Ca2+ responses elicited by 10 μM 5-hydroxytryptamine (serotonin) in canine PASMCs and 100 μM carbachol in human embryonic kidney (HEK)-293 cells were not reduced by 2 μM FLA 365, but they were reduced by 20 μM FLA 365 to 76 ± 9% of control in canine PASMCs and 52 ± 1% in HEK-293 cells. Thus, FLA 365 preferentially blocks RyRs with limited inhibition of L-type Ca2+ channels or InsP 3R in canine PASMCs. Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics.