Ostrovskaya Olga, Goyal Ravi, Osman Noah, McAllister Claire E, Pessah Isaac N, Hume Joseph R, Wilson Sean M
Department of Pharmacology, University of Mississippi School of Pharmacy, University of Mississippi, 303 Faser Hall, University, MS 38677, USA.
J Pharmacol Exp Ther. 2007 Oct;323(1):381-90. doi: 10.1124/jpet.107.122119. Epub 2007 Jul 19.
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.
雷诺丁是一种选择性雷诺丁受体（RyR）阻滞剂，其结合依赖于RyR的开放。在全细胞研究中，雷诺丁结合可将RyR锁定在开放导电状态，使肌浆网短路，这限制了对肌醇-1,4,5-三磷酸受体（InsP3R）活性的研究。其他RyR阻滞剂具有非选择性作用，这也限制了它们的用途。4-（2-氨基丙基）-3,5-二氯-N,N-二甲基苯胺（FLA 365）可阻断RyR引起的骨骼肌和心肌中Ca2+的增加；然而，其对平滑肌的作用尚不清楚。犬肺动脉平滑肌细胞（PASMCs）同时表达RyRs和InsP3Rs；因此，我们通过对负载fura-2的PASMCs进行成像，测试了FLA 365阻断RyR和5-羟色胺介导的InsP3R引起的Ca2+释放的能力。急性暴露于10 mM咖啡因（一种选择性RyR激活剂）可诱导Ca2+增加，而FLA 365可使其可逆性降低，估计IC50约为1至1.5 microM，且可被10 microM雷诺丁或10 microM环匹阿尼酸抑制。FLA 365还可阻断L型Ca2+通道活性，在膜片钳电压钳研究中，10 microM可使Ba2+电流幅度降至对照的54±6%，100 microM FLA 365可使膜电流降至21±6%。2 microM FLA 365不会降低犬PASMCs中由10 microM 5-羟色胺（5-羟色胺）和人胚肾（HEK）-293细胞中由100 microM卡巴胆碱引起的InsP3R介导的Ca2+反应，但在犬PASMCs中，20 microM FLA 365可使其降至对照的76±9%，在HEK-293细胞中降至52±1%。因此，在犬PASMCs中，FLA 365优先阻断RyRs，对L型Ca2+通道或InsP3R的抑制作用有限。
