Background and purpose: Experiments were performed to determine if capacitative Ca 2 þ entry (CCE) in canine pulmonary arterial smooth muscle cells (PASMCs) is dependent on InsP 3 receptors or ryanodine receptors as induction of CCE is dependent on simultaneous depletion of the functionally separate InsP 3 -and ryanodine-sensitive sarcoplasmic reticulum (SR) Ca 2 þ stores in these cells. Experimental approach: Myocytes were isolated from canine pulmonary arteries using enzymatic procedures and were used within 8 h of preparation. Measurements of cytosolic Ca 2 þ were made by imaging fura-2 loaded individual myocytes that were perfused with physiological buffered saline solution with or without Ca 2 þ .

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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 3 R) 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 Ca 2ϩ 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 InsP 3 Rs; thus, we tested the ability of FLA 365 to block RyR-and serotonin-mediated InsP 3 R-elicited Ca 2ϩ release by imaging fura-2-loaded PASMCs. Acute exposure to 10 mM caffeine, a selective RyR activator, induced Ca 2ϩ increases that were reversibly reduced by FLA 365, with an estimated IC 50 of ϳ1 to 1.5 M, and inhibited by 10 M ryanodine or 10 M cyclopiazonic acid. FLA 365 also blocked L-type Ca 2ϩ channel activity, with 10 M reducing Ba 2ϩ current amplitude in patch voltage-clamp studies to 54 Ϯ 6% of control and 100 M FLA 365 reducing membrane current to 21 Ϯ 6%. InsP 3 R-mediated Ca 2ϩ responses elicited by 10 M 5-hydroxytryptamine (serotonin) in canine PASMCs and 100 M carbachol in human embryonic kidney (HEK)-293 cells were not reduced by 2 M FLA 365, but they were reduced by 20 M FLA 365 to 76 Ϯ 9% of control in canine PASMCs and 52 Ϯ 1% in HEK-293 cells. Thus, FLA 365 preferentially blocks RyRs with limited inhibition of L-type Ca 2ϩ channels or InsP 3 R in canine PASMCs.

Caffeine is a well described and characterized ryanodine receptor (RyR) activator. Previous evidence from independent research studies also indicate caffeine inhibits InsP 3 receptor functionality, which is important to activation of capacitative Ca 2+ entry (CCE) in some cell types. In addition, RyR activation elicits excitatory-coupled Ca 2+ entry (ECCE) in skeletal muscle myotubes. Recent studies by our group show that canine pulmonary arterial smooth muscle cells (PASMCs) have functional InsP 3 receptors as well as RyRs, and that CCE is dependent on InsP 3 receptor activity. The potential for caffeine to activate ECCE as well as inhibit InsP 3 receptor function and CCE was examined using fura-2 fluorescent imaging in canine PASMCs. The data show caffeine causes transient as well as sustained cytosolic Ca 2+ increases, though this is not due to CCE or ECCE activity as evidenced by a lack of an increase in Mn 2+ quench of fura-2. The experiments also show caffeine reversibly inhibits 5-HT elicited -InsP 3 mediated Ca 2+ responses with an IC 50 of 6.87 × 10 −4 M and 10 mM caffeine fully inhibits CCE. These studies provide the first evidence that caffeine is an inhibitor of InsP 3 generated Ca 2+ signals and CCE in PASMCs.

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