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

Olga Ostrovskaya, Ravi Goyal, Noah Osman, Claire E. McAllister, Isaac N Pessah, Joseph R. Hume, Sean M. Wilson

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)381-390
Number of pages10
JournalJournal of Pharmacology and Experimental Therapeutics
Volume323
Issue number1
DOIs
StatePublished - Oct 2007

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Ryanodine Receptor Calcium Release Channel
Smooth Muscle Myocytes
Canidae
Lung
Ryanodine
Serotonin
Kidney
Inositol 1,4,5-Trisphosphate Receptors
N,N-dimethylaniline
FLA 365
Fura-2
Sarcoplasmic Reticulum
Carbachol
Caffeine
Inhibitory Concentration 50
Smooth Muscle
Myocardium
Skeletal Muscle
Membranes

ASJC Scopus subject areas

  • Pharmacology

Cite this

Inhibition of ryanodine receptors by 4-(2-aminopropyl)-3,5-dichloro-N,N- dimethylaniline (FLA 365) in canine pulmonary arterial smooth muscle cells. / Ostrovskaya, Olga; Goyal, Ravi; Osman, Noah; McAllister, Claire E.; Pessah, Isaac N; Hume, Joseph R.; Wilson, Sean M.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 323, No. 1, 10.2007, p. 381-390.

Research output: Contribution to journalArticle

Ostrovskaya, Olga ; Goyal, Ravi ; Osman, Noah ; McAllister, Claire E. ; Pessah, Isaac N ; Hume, Joseph R. ; Wilson, Sean M. / Inhibition of ryanodine receptors by 4-(2-aminopropyl)-3,5-dichloro-N,N- dimethylaniline (FLA 365) in canine pulmonary arterial smooth muscle cells. In: Journal of Pharmacology and Experimental Therapeutics. 2007 ; Vol. 323, No. 1. pp. 381-390.
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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.",
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