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CARDIOVASCULAR

Inhibition of Ryanodine Receptors by 4-(2-Aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365) in Canine Pulmonary Arterial Smooth Muscle Cells

Department of Pharmacology, University of Mississippi School of Pharmacy and Research Institute of Pharmaceutical Sciences, University, Mississippi (O.O., R.G., N.O., S.M.W.); University of Mississippi Light Microscopy Core, University, Mississippi (N.O., S.M.W.); Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada (C.E.M., J.R.H.); and Department of Molecular Biosciences, School of Veterinary Sciences, University of California, Davis, California (I.N.P.)

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 InsP₃ R-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 1 to 1.5 µM, and inhibited by 10 µM ryanodine or 10 µM cyclopiazonic acid. FLA 365 also blocked L-type Ca²⁺ 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%. InsP₃R-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 InsP₃R in canine PASMCs.

Address correspondence to: Dr. Sean M. Wilson, University of Mississippi School of Pharmacy, University of Mississippi, 303 Faser Hall, University, MS 38677. E-mail: wilson{at}olemiss.edu