title: 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 (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.
authors.affiliation: Department of Pharmacology, University of Mississippi School of Pharmacy, University of Mississippi, 303 Faser Hall, University, MS 38677, USA.
authors.forename: Olga
authors.initials: O
authors.lastname: Ostrovskaya
authors.forename: Ravi
authors.initials: R
authors.lastname: Goyal
authors.forename: Noah
authors.initials: N
authors.lastname: Osman
authors.forename: Claire E
authors.initials: CE
authors.lastname: McAllister
authors.forename: Isaac N
authors.initials: IN
authors.lastname: Pessah
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Hume
authors.forename: Sean M
authors.initials: SM
authors.lastname: Wilson
mesh_terms: D000818:Animals; D001464:Barium / Q000378:metabolism; D002110:Caffeine / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D005260:Female; D007211:Indoles / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D010627:Phenethylamines / Q000494:pharmacology*; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*
chemical_list: D002121:Calcium Channel Blockers; D007211:Indoles; D010627:Phenethylamines; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D001464:Barium; D002110:Caffeine; C060909:FLA 365; C000543:cyclopiazonic acid