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

• Published in: The Journal of pharmacology and experimental therapeutics Vol. 323; no. 1; p. 381
• Main Authors: Ostrovskaya, Olga, Goyal, Ravi, Osman, Noah, McAllister, Claire E, Pessah, Isaac N, Hume, Joseph R, Wilson, Sean M
• Format: Journal Article
• Language: English
• Published: United States 01-10-2007
• Subjects: Animals; Barium - metabolism; Caffeine - pharmacology; Calcium Channel Blockers - pharmacology; Calcium Signaling - drug effects; Cell Line; Dogs; Dose-Response Relationship, Drug; Electrophysiology; Female; Indoles - pharmacology; Male; Muscle, Smooth, Vascular - cytology; Muscle, Smooth, Vascular - drug effects; Muscle, Smooth, Vascular - metabolism; Myocytes, Smooth Muscle - drug effects; Myocytes, Smooth Muscle - metabolism; Phenethylamines - pharmacology; Pulmonary Artery - cytology; Pulmonary Artery - drug effects; Pulmonary Artery - metabolism; Ryanodine - pharmacology; Ryanodine Receptor Calcium Release Channel - metabolism
• ISSN: 0022-3565
• DOI: 10.1124/jpet.107.122119

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 (InsP3R) 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 InsP3R-elicited Ca2+ release by imaging fura-2-loaded PASMCs. Acute exposure to 10 mM caffeine, a selective RyR activator, induced Ca2+ increases that were reversibly reduced by FLA 365, with an estimated IC50 of approximately 1 to 1.5 microM, and inhibited by 10 microM ryanodine or 10 microM cyclopiazonic acid. FLA 365 also blocked L-type Ca2+ channel activity, with 10 microM reducing Ba2+ current amplitude in patch voltage-clamp studies to 54 +/- 6% of control and 100 microM FLA 365 reducing membrane current to 21 +/- 6%. InsP3R-mediated Ca2+ responses elicited by 10 microM 5-hydroxytryptamine (serotonin) in canine PASMCs and 100 microM carbachol in human embryonic kidney (HEK)-293 cells were not reduced by 2 microM FLA 365, but they were reduced by 20 microM 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 InsP3R in canine PASMCs.