FLA 365 as an inhibitor of ryanodine receptors and L‐type Ca 2+ channels in arterial myocytes

Abstract

Ryanodine (RY) is a highly selective ryanodine receptor (RyR) blocker, but RY binding is dependent on RyR opening. In whole-cell studies, RY binding can lock the RyR in an open-conductance state, short-circuiting the sarcoplasmic reticulum (SR). This restricts studies of InsP3 receptor (InsP3R) activity. Other RyR blockers also have non-selective effects that also limit their utility. FLA 365 ([2,6-dichloro-4-dimethyl-aminophenyl] isopropylamine) 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 (FLA) to block RyR and 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 significantly reduced by 20 μM FLA, which was reversible. 10–100 μM FLA 365 reduced Ba2+ currents through L-type Ca2+ channels in patch voltage-clamp studies, similar to phenylalkylamines. 10 μM 5-HT, which activates InsP3Rs, induced Ca2+ increases of equivalent amplitude in the absence or presence of FLA. Thus, FLA blocks RyRs and L-type Ca2+ channels with limited impact on InsP3R signaling in PASMCs. NIH P20RR15518 from NCRR & HL49254 (JRH), PO1ES11269 & 2PO AR17605 (INP), HL10476, AI55462 & UM faculty fellowship (SMW).