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 (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.
Footnotes
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This work was supported by National Institutes of Health Grants HL49254 and P20RR15518 from National Center for Research Resources (to J.R.H.); Grants ES11269 and AR17605 (to I.N.P.); Grants HL10476 and AI55642; and a University of Mississippi faculty fellowship. A portion of this material is based upon work supported by the National Science Foundation under Grant MRI 0619774 (to S.M.W.), a University of Mississippi graduate student fellowship (to R.G.), and by the National Center for Natural Product Research.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.122119.
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ABBREVIATIONS: RyR, ryanodine receptor; SR, sarcoplasmic reticulum; PASMC, pulmonary arterial smooth muscle cell; InsP3, inositol-1,4,5-trisphosphate; InsP3R, inositol-1,4,5-trisphosphate receptor; Cav, calcium channel; RuR, ruthenium red; FLA 365, 4-(2-aminopropyl)-3,5-dichloro-N,N-dimethylaniline; PSS, physiological saline solution; HEK, human embryonic kidney; AM, acetoxymethyl ester; IBa, Ba2+ current; 5-HT, 5-hydroxytryptamine (serotonin); CCh, carbachol; ANOVA, analysis of variance; SERCA, sarcoplasmic-endoplasmic reticulum Ca2+ ATPase; Rya, ryanodine; CPA, cyclopiazonic acid; CAF, caffeine.
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↵1 Current affiliation: Department of Pharmacology University of Tennessee, Memphis, Tennessee.
- Received March 5, 2007.
- Accepted July 18, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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