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CARDIOVASCULAR

Inhibition of Ryanodine Receptors by 4-(2-Aminopropyl)-3,5-dichloro-N,N-dimethylaniline (FLA 365) in Canine Pulmonary Arterial Smooth Muscle Cells

Department of Pharmacology, University of Mississippi School of Pharmacy and Research Institute of Pharmaceutical Sciences, University, Mississippi (O.O., R.G., N.O., S.M.W.); University of Mississippi Light Microscopy Core, University, Mississippi (N.O., S.M.W.); Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada (C.E.M., J.R.H.); and Department of Molecular Biosciences, School of Veterinary Sciences, University of California, Davis, California (I.N.P.)

Received March 5, 2007; accepted July 18, 2007.

	Abstract

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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 (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.

The nomenclature of RyRs is due to the fact that the plant alkaloid ryanodine binds with high selectivity to this ion channel. Yet, ryanodine binding is dependent on RyR opening, with both concentration- and time-dependent effects on channel gating. Low ryanodine concentrations or short exposure periods lock the channel into a subconductance state, whereas high ryanodine concentrations and long exposure times fully block the channel (Pessah and Zimanyi, 1991). Thus, in whole-cell studies of smooth muscle ryanodine often locks the RyR into an open subconductance state, which then leads to depletion of the SR Ca²⁺ stores (Janiak et al., 2001; Wilson et al., 2002). Ryanodine-mediated depletion of the SR Ca²⁺ stores then may activate store-operated Ca²⁺ influx pathways, which could influence data interpretation (Wilson et al., 2002). Given that the inositol-1,4,5-trisphosphate receptor (InsP₃R) may also be on contiguous membrane with RyRs, it can be difficult to study the InsP₃R or RyR activation in isolation or potential interactions between the two SR Ca²⁺ release channels. Because of this, there is a need for potent RyR antagonists that have little or no effect on other aspects of intracellular Ca²⁺ homeostasis.

A number of compounds are commonly used as RyR channel blockers, but like ryanodine, their use can also be problematic. The polycationic dye ruthenium red (RuR) is a well known RyR channel blocker (Smith et al., 1988; Ma, 1993; Chen and MacLennan, 1994). However, it can block voltagegated Ca²⁺ channels (Ca_V) (Cibulsky and Sather, 1999), several transient receptor potential channel isoforms (Bleakman et al., 1990; Dray et al., 1990; Nagata et al., 2005), K⁺ channels (Wann and Richards, 1994; Lin and Lin-Shiau, 1996; Hirano et al., 1998), and Ca²⁺-binding proteins (Charuk et al., 1990). RuR also alters the Ca²⁺ uptake and release properties of mitochondria (Rossi et al., 1973). Neomycin affects RyR similarly to RuR, but it, too, blocks Ca_V (Canzoniero et al., 1993) as well as ATP-activated K⁺ channels (Lin et al., 1993), and phospholipase C (Wang et al., 2005). The anesthetic tetracaine is a well used RyR inhibitor, but importantly, it inhibits InsP₃R activity (MacMillan et al., 2005). FLA 365 was originally designed as a monamine oxidase inhibitor (Ask et al., 1985; Ask and Ross, 1987). FLA 365 also reduces RyR-elicited Ca²⁺ release from the SR of skeletal and cardiac muscle (Chiesi et al., 1988; Calviello and Chiesi, 1989; Mack et al., 1992), and it inhibits ryanodine binding (Mack et al., 1992); yet, the actions of FLA 365 on smooth muscle Ca²⁺ signaling are unknown. Canine PASMCs isolated from pulmonary resistance arteries have well described caffeine-ryanodine and InsP₃ -sensitive Ca²⁺ release stores (Janiak et al., 2001), making this an excellent arterial smooth muscle model for pharmaceutical studies involving SR Ca²⁺ metabolism. The present series of experiments take advantage of the SR Ca²⁺ store properties to test the hypothesis that FLA 365 inhibits RyR function in arterial smooth muscle.

	Materials and Methods

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Cell Isolation. Smooth muscle cells were isolated from high-resistance canine pulmonary arteries as described previously (Janiak et al., 2001; Wilson et al., 2002). Mongrel dogs of either sex were sacrificed with pentobarbital sodium (45 mg kg^–1 i.v.) and ketamine (15 mg kg^–1 i.v.), as approved by the University of Nevada at Reno Institutional Animal Care and Use Committee. The heart and lungs were excised en bloc. The third and fourth branches of pulmonary arteries were dissected at 5°C to decrease cellular metabolic activity. Pulmonary artery isolations and smooth muscle cell dispersions were made in a low-Ca²⁺ physiological saline solution (PSS) containing 125 mM NaCl, 5.36 mM KCl, 0.336 mM Na₂HPO₄, 0.44 mM K₂HPO₄, 11 mM HEPES, 1.2 mM MgCl₂, 0.05 mM CaCl₂, and 10 mM glucose, pH 7.4 (adjusted with Tris), with osmolarity 300 mOsm. Arteries were cleaned of connective tissue, cut into small pieces, and placed in a tube containing fresh PSS. Tissue was immediately digested or stored at 5°C up to 24 h. To disperse cells, tissue was placed in low-Ca²⁺ PSS containing the following enzymes: 0.5 mg ml^–1 collagenase type XI; 0.03 mg ml^–1 elastase type IV, and 0.5 mg ml^–1 bovine serum albumin (fat-free) for 14 to 16 h at 5°C. In many cases, tissues in digestion solution were shipped overnight from the University of Nevada (Reno, NV) to the University of Mississippi (University, MS) at 5°C. The tissue was then washed several times with 5°C low-Ca²⁺ PSS solution and triturated with a fire-polished Pasteur pipette. The resulting dispersed PASMCs were cold stored at 5°C up to 8 h until experiments were performed.

Cell Culture. HEK-293 cells obtained from American Type Culture Collection (Manassas, VA) were cultured at 37°C in Eagle's modified essential medium containing 10% fetal bovine serum in 5% CO₂. For Ca²⁺ measurements, cells were plated on glass coverslips (Corning Glassworks, Corning, NY) and used within 48 to 72 h after plating.

Fluorescence Imaging. The cytosolic [Ca²⁺] was measured in canine PASMCs or HEK-293 cells loaded with the ratiometric Ca²⁺-sensitive dye fura-2 acetoxymethyl ester (AM) (Invitrogen, Carlsbad, CA) using a dual excitation digital Ca²⁺ imaging system (IonOptix Inc., Milton, MA) equipped with an intensified charge-coupled device. The imaging system was mounted on a TS100 inverted microscope (Nikon, Melville, NY) outfitted with a 40x (numerical aperture 1.3; Nikon) oil immersion objective. The fura-2 AM was dissolved in dimethyl sulfoxide and added from a 1 mM stock to the PASM cell suspension or HEK-293 cells attached to coverslips at a final concentration of 10 µM. Cells were loaded with fura-2 AM for 20 to 30 min in a perfusion chamber (Warner Instruments, Hamden, CT) at room temperature in the dark. Cells were then washed for 30 min to allow for dye esterification at 1 ml min^–1 with a balanced salt solution of the following composition: 126 mM NaCl, 5 mM KCl, 0.3 mM NaH₂PO₄, 10 mM HEPES, 1 mM MgCl₂, 2 mM CaCl₂, and 10 mM glucose, pH 7.4 (adjusted with NaOH), with osmolarity 285 to 295 mOsm. Cells were continuously perfused with a peristaltic pump (Rainin Instruments, Woburn, MA or Masterflex Cole-Parmer Instrument Co., Vernon Hills, IL), and solution flow was controlled with a multichannel ValveBank computerized system connected to pinch valves (Automate Scientific, Berkeley, CA). Measurements of cytosolic [Ca²⁺] before and during pharmacological manipulation were made once the fura-2 fluorescence ratio stabilized. Cells were illuminated with a xenon arc lamp at 340 and 380 nm (Chroma Technology Corp., Rockingham, VT) and emitted light was collected from regions that encompassed single cells with a charge-coupled device at 510 nm. In most experiments, images were acquired at 1 Hz and stored on either compact disk or magnetic media for later analysis. Although it is difficult to precisely measure the intracellular calcium concentration ([Ca²⁺]_i) (Baylor and Hollingworth, 2000), estimates were made from the relation [Ca²⁺]_i = K_d x (Sf₂/Sb₂) x (R – R_min)/(R_max – R), where R_min and R_max are the F₃₄₀/F₃₈₀ ratios of Ca²⁺-free and Ca²⁺-saturated fura-2, respectively. Sf₂ is the F₃₈₀ of Ca²⁺-free fura-2, and Sb₂ is F₃₈₀ of Ca²⁺-bound fura-2. The values of Sf₂ and R_min were determined by bathing cells in a balanced salt solution that did not have any added Ca²⁺ and contained 10 mM EGTA and 1 µM ionomycin. The values of Sb₂ and R_max were determined by bathing cells in a balanced salt solution that contained 10 mM Ca²⁺ and 1 µM ionomycin. The K_d for fura-2 was assumed to be 224 nM (Grynkiewicz et al., 1985). Experimental temperature was 22–25°C.

Electrophysiology. Ba²⁺ currents (I_Ba) through L-type Ca²⁺ channels were measured using the dialyzed whole-cell configuration of the patch voltage-clamp technique (Hamill et al., 1981). The voltage protocol used to record I_Ba consisted of a holding potential of –80 mV, and cells were depolarized with 100-ms pulses to +10 mV once every 3 s. The capacitance was directly read from the HEKA EPC10 amplifier (HEKA Instruments Inc., Southboro, MA) once the cell capacitance was compensated. Micropipettes were pulled from glass capillaries (BF 150-86-10; Sutter Instrument Company, Novato, CA) with a Sutter P97 horizontal pipette puller and had tip resistances in the range of 2 to 5 M. Series resistance was compensated 50 to 80% if needed to give a final value below 10 M. Amplified currents were acquired through a LIH-1600 (HEKA Instruments Inc.) computer interface card and filtered through two filters, with the first filter being 10 KHz and the second filter 2.9 being KHz. Data were acquired with Patchmaster version 2.1 (HEKA Instruments Inc.), and they were stored on magnetic media and compact disk for offline analysis.

Cells were perfused during all experiments with an external solution using a gravity perfusion system at 1 ml/min, and solution flow was controlled with a multichannel ValveLink electronically controlled system connected to pinch valves (Automate Scientific, Berkeley, CA). The external solution had the following composition: 118 mM NaCl, 5 mM CsCl, 10 mM HEPES, 1 mM MgCl₂, 10 mM glucose, and 10 mM BaCl₂·2H₂O, pH 7.4 (adjusted with NaOH), with osmolarity 280 to 290 mOsm. The pipette solution was 118 mM CsCl, 10 mM tetraethylammonium-chloride, 10 mM EGTA, 10 mM HEPES, 0.1 mM GTP, 5 mM ATPNa₂, and 2 mM MgCl₂, pH7.3 (adjusted with CsOH), 280 to 290 mOsm, as determined with an osmometer (model 5520; Wescor, Logan, UT).

Chemicals and Drugs. FLA 365 was provided by I. N. Pessah (Department of Molecular Biosciences, School of Veterinary Sciences, University of California, Davis, CA). Ionomycin free acid (C₄₁H₇₀O₉) was purchased from Calbiochem (San Diego, CA); fura-2 AM was from Invitrogen; 5-hydroxytryptamine (serotonin, 5-HT), 2-carbamoyloxyethyl-trimethyl-azanium chloride (carbachol; CCh), ryanodine (C₂₅H₃₅NO₉), cyclopiazonic acid (C₂₀H₂₀N₂O₃), 1,3,7-trimethylxanthine (caffeine), 2-(3,4-dimethoxyphenyl)-5-[2-(3,4-dimethoxyphenyl)ethyl-methyl-amino]-2-(1-methylethyl) pentanenitrile (verapamil), dimethyl2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (nifedipine), and all other chemicals purchased from Sigma-Aldrich (St. Louis, MO).

Statistical Analysis. All data are presented as mean ± S.E.M. Statistical difference within cell groups was determined with a two-tailed paired Student's t test. Statistical tests between groups were performed with analysis of variance (ANOVA) with the specific test being chosen dependent on the type of samples being examined, normality of the data set, or the variability within or between groups. A Kruskal-Wallis one-way ANOVA on ranks with a Dunn's pairwise multiple comparison procedure was used if the data distribution did not pass normality. A one-way ANOVA with a Newman-Keuls or a Bonferroni's multiple comparison test was used for comparing different independent cell groups. A repeated measures ANOVA with a Newman-Keuls multiple comparison test was used to test the difference between the effects of different conditions in the same cell group. The specific test used for each data set is noted in the legend for each figure. A P value <0.05 was accepted as statistically significant. The n values reported reflect the total number of cells tested. For the dose-response curve depicted in Fig. 1B, the following number of cells were examined and treated with the following concentrations of FLA 365: 20 cells were not exposed to FLA 365; 9 cells were treated with 1 nM FLA 365, 20 cells with 10 nM FLA 365, 14 cells with 1 µM FLA 365, 21 cells with 2 µM FLA 365, 11 cells with 10 µM FLA 365, 24 cells with 20 µM FLA 365, 7 cells with 50 µM FLA 365, and 11 cells with 200 µM FLA 365. Multiple trials were performed on cells isolated from multiple dogs for most experimental paradigms. When HEK-293 cells were used, at least three independent experimental runs were performed.

View larger version (9K): [in this window] [in a new window]   Fig. 1. FLA 365 causes a concentration-dependent inhibition of caffeineelicited cytosolic Ca2+ increases in canine PASMCs. A, representative tracing of caffeine (CAF) induced Ca2+ increases in the absence and presence of 2 µM and then 200 µM FLA 365. B, average change in the F340/F380 response to caffeine compared with their respective control values for varied FLA 365 concentrations. Data were fit with a Hill equation exclusive (solid line) or inclusive (dashed line) of 20 µM FLA 365 (open circle). Error bars represent ± S.E.M.

	Results

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FLA 365 Causes a Reversible Inhibition of RyR in Canine PASMCs. Canine PASMCs have caffeine-ryanodine- and InsP₃ -sensitive SR Ca²⁺ stores (Janiak et al., 2001), which makes them an excellent model system for pharmacological examinations of compounds that may effect RyR or InsP₃R activity. Figure 1A shows the Ca²⁺ response to 10 mM caffeine in an individual canine PASMC and the blocking action of two concentrations of FLA 365. Exposure of the cell to 2 µM FLA 365 did not alter the cytosolic Ca²⁺ concentration on its own, but it reduced the Ca²⁺ response to 10 mM caffeine by 75%. Elevating FLA 365 to 200 µM induced a transient increase in the cytosolic Ca²⁺ concentration. Cells treated with FLA 365 concentrations 20 µM also had Ca²⁺ elevations and are presented in Fig. 4. Exposure to 10 mM caffeine in the continued presence of 200 µM FLA 365 did not cause any cytosolic Ca²⁺ increase.

View larger version (9K): [in this window] [in a new window]   Fig. 4. FLA 365 elicits cytosolic Ca2+ elevations in some canine PASMCs. A, effect of 20 µM FLA 365 on the cytosolic [Ca2+] in a responsive cell. B, bars show the cytosolic [Ca2+] in the absence and then presence of 20 µM FLA 365. **, significant difference from control by a two-tailed paired t test (P < 0.01). C, bars show the percentage of cells with (responders; open bars) or without (nonresponders; solid bars) cytosolic Ca2+ elevations in response to 20 µM FLA 365 (n = 48). Number in parentheses is the number of cells examined. Error bars represent ±S.E.M.

For comparative purposes and to show some of the potential utility of FLA 365, a series of experiments were performed where 10 µM ryanodine or 10 µM cyclopiazonic acid, a sarcoplasmic-endoplasmic reticulum Ca²⁺ ATPase (SERCA) inhibitor, were used to inhibit caffeine-elicited RyR-mediated Ca²⁺ responses. Figure 2A shows that 10 mM caffeine applications repeated every 3 min elicited Ca²⁺ responses of similar magnitude in an individual canine PASMC. Figure 2B then shows an individual myocyte exposed to 10 µM ryanodine, where the Ca²⁺ response to 10 mM caffeine decayed with sequential stimulations. Figure 2C shows that 10 µM cyclopiazonic acid induced Ca²⁺ elevations on its own, corresponding to SERCA inhibition and passive loss of Ca²⁺ from the SR. Cyclopiazonic acid also reduced and ablated the responses to 10 mM caffeine through the loss of stored Ca²⁺. Figure 2D summarizes the changes in the magnitude of the Ca²⁺ release events with repeated caffeine applications in the absence and presence of 10 µM ryanodine or 10 µM cyclopiazonic acid. The figure illustrates that the Ca²⁺ response to 10 mM caffeine is reduced in the presence of ryanodine, with the second caffeine response being 73 ± 11% of the control and 34 ± 5% with a third caffeine treatment. Cyclopiazonic acid similarly reduced the Ca²⁺ elevations due to caffeine, where the second caffeine response was 27 ± 3% of the control and 2 ± 1% when treated with caffeine once more. These ryanodine and cyclopiazonic acid-dependent decrements in Ca²⁺ responsiveness to caffeine are similar to the results that we have published previously (Janiak et al., 2001; Wilson et al., 2002). Comparatively, in cells that were not treated with ryanodine or cyclopiazonic acid, there were not any decreases in the Ca²⁺ responses to sequential applications of caffeine. The second caffeine application elicited a Ca²⁺ response that was 119 ± 8% of the first caffeine exposure (i.e., control), whereas a third application was 142 ± 8%.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Ryanodine or cyclopiazonic acid inhibit caffeine elicited cytosolic Ca2+ increases in canine PASMCs. Representative tracings of repetitive caffeine-induced Ca2+ increases in the absence (A) and presence (B) of ryanodine (Rya) or cyclopiazonic acid (CPA; C). D, bars indicate the percentage of the F340/F380 for 10 mM caffeine for the second and third caffeine exposure compared with the first exposure in the absence (solid) or presence of 10 µM Rya (open) or 10 µM CPA (diagonal lines). Significant differences between the responses in the presence and absence of Rya or CPA are denoted for the second and third caffeine applications. *, P < 0.05 and ***, P < 0.001 by a Kruskal-Wallis one-way analysis of variance on ranks with a Dunn's pairwise multiple comparison procedure. Number in parentheses is the number of cells examined. Error bars represent ± S.E.M.

View larger version (9K): [in this window] [in a new window]   Fig. 3. FLA 365 reversibly inhibits caffeine-elicited cytosolic Ca2+ increases in canine PASMCs. A, representative tracing of caffeine-induced Ca2+ increases in the absence, presence, and following washout of 20 µM FLA 365. B, bars show the change in cytosolic [Ca2+] from the resting [Ca2+] due to 10 mM caffeine in the absence, presence, and following washout of 20 µM FLA 365. *, significantly different by Kruskal-Wallis one-way analysis of variance on ranks with a Dunn's pairwise multiple comparison procedure from CAF and CAF (Wash) groups (P < 0.05). Number in parentheses is the number of cells examined. Error bars represent ± S.E.M.

FLA 365 is known to cause rapid inhibition of RyR activity (Chiesi et al., 1988; Calviello and Chiesi, 1989; Mack et al., 1992), although there may be a short latency between FLA 365 binding and RyR block. During this time, the RyR may be in a longer lived open state, which would allow for significant Ca²⁺ flux, such as occurs with ryanodine binding. To test this, we examined the actions of FLA 365 on resting myocytes. Figure 4A shows a representative cell where 20 µM FLA 365 did cause a significant increase in the cytosolic [Ca²⁺]. Figure 4B summarizes the Ca²⁺ responses for the FLA 365-responsive myocytes, illustrating that 20 µM FLA 365 caused the cytosolic [Ca²⁺] to rise 100 nM, from 128 ± 19 to 228 ± 25 nM in the eight cells where FLA 365 induced Ca²⁺ increases. Figure 4C illustrates that Ca²⁺ elevations due to 20 µM FLA 365 are infrequent, occurring in only 8 of 48 myocytes (16.7%). Notably cells exposed to >20 µM FLA 365 also had cytosolic Ca²⁺ increases as illustrated for 200 µM FLA 365 in Fig. 1A, whereas those exposed to 2 µMor lower concentrations did not.

FLA 365 blocks L-Type Ca Channels in PASMCs. FLA 365 is a phenylalkylamine; thus, it belongs to the same major chemical class as verapamil, which is a potent and selective blocker of L-type Ca²⁺ channels (Catterall et al., 2005). Because of this, we tested the hypothesis that FLA 365 would reduce Ca_V function in PASMCs. To assess the function of Ca_V, Ba²⁺ currents were measured using whole-cell patch voltage-clamp techniques (del Corsso et al., 2006). Figure 5A shows the percentage of the peak Ba²⁺ current in an individual myocyte held at –80 mV and stepped to +10 mV every 3 s, which elicits Ca_V activity. In this cell, there was very little rundown of the current amplitude over time, and, on average, there was only a modest rundown of the membrane current, being 92 ± 3% of the initial value (Fig. 5D). Figure 5B shows that 10 µM verapamil caused the Ba²⁺ current to be reduced to 25% of the initial amplitude. On average, 10 µM verapamil caused the Ba²⁺ current amplitude to be reduced to 15 ± 6%. The effects of FLA 365 on voltage-activated Ba²⁺ currents were then examined at 10 and 100 µM, which are well above the IC₅₀ for FLA 365 inhibition of the RyR. Figure 5C illustrates that 100 µM FLA 365 caused a reduction in the Ba²⁺ current amplitude, which is comparable with that of 10 µM verapamil. Figure 5D shows that, on average, Ba²⁺ currents were reduced to 54 ± 6% of the control current by 10 µM FLA 365, whereas 100 µM FLA 365 reduced the current to 21 ± 6% compared with control. Thus, FLA 365 exerts a significant block on I_Ba in canine PASMCs, with an approximated IC₅₀ of 10 µM, which is about 1 order of magnitude higher than the IC₅₀ of RyR block by FLA 365 (Fig. 1).

View larger version (17K): [in this window] [in a new window]   Fig. 5. FLA 365 blocks IBa in canine PASMCs. A, Ba2+ currents over the same time period as cells treated with various CaV antagonists. Effects of 10 µM verapamil (B) or 100 µM FLA 365 (C) on IBa. D, bars indicate the percentage of IBa remaining under time-matched control conditions or in response to 10 µM verapamil or 10 µM or 100 µM FLA 365. Black circles and inset traces show current under control conditions, whereas gray circles and traces show current in response to treatments illustrated in each panel. ***, P < 0.001, significant difference from control. , P < 0.01, significant difference from 10 µM FLA by a one-way ANOVA with a Newman-Keuls multiple comparison test. Number in parentheses is the number of cells examined. Error bars represent ± S.E.M.

View larger version (16K): [in this window] [in a new window]   Fig. 6. CaV inhibition does not reduce caffeine or 5-HT mediated Ca2+ responses in canine PASMCs. A, representative tracing of the Ca2+ responses to repeated 10 mM caffeine applications. B, effects of 10 µM verapamil (Vera) on caffeine-mediated Ca2+ responses. C, effects of 10 µM nifedipine (NIF) on 10 µM 5-HT induced Ca2+ responses. D, bars indicate the percentage of the F340/F380 relative to an initial application of 10 mM caffeine (solid bars) or 10 µM 5-HT (open bars) in the presence of the agents listed. ***, P < 0.001, significant difference to other groups treated with caffeine by a one-way ANOVA with a Newman-Keuls multiple comparison test. Number in parentheses is the number of cells examined. Error bars represent ± S.E.M.

FLA 365 Reduces 5-HT-Mediated Ca²⁺ Responses in Canine PASMCs. Even though FLA 365 has long been known to inhibit RyR activity, its actions on InsP₃ related Ca²⁺ release events have not been previously examined. This question was explored by testing the actions of FLA 365 on 5-HT-elicited Ca²⁺ responses in canine PASMCs. The 5-HT Ca²⁺ responses in canine pulmonary arterial myocytes are due to the activity of InsP₃ receptors, because the responses are blocked by ketanserin, a selective 5-HT_2A receptor antagonist as well as by the InsP₃ receptor blockers 2-aminobiphenylborate and xestospongin C (Wilson et al., 2005). Figure 7A as well as Fig. 6C show that 5-HT exposures repeated 5 min can induce Ca²⁺ responses of similar magnitude. Figure 7B shows that when treated with 2 µM FLA 365, the amplitude of the 5-HT-elicited Ca²⁺ response is unaffected, whereas 10 mM caffeine-elicited Ca²⁺ responses are significantly depressed. Figure 7C illustrates that 20 µM FLA 365 reduces the amplitude of the 5-HT-elicited Ca²⁺ response by 25% and that it reduces 10 mM caffeine-elicited Ca²⁺ increases substantially more.

View larger version (15K): [in this window] [in a new window]   Fig. 7. FLA 365 reduces 5-HT induced Ca2+ responses in canine PASMCs. A, representative tracing of the Ca2+ responses to repeated 10 µM 5-HT applications. B, effects of 2 µM FLA 365 (B) and 20 µM FLA 365 (C) on 10 mM caffeine as well as 10 µM 5-HT-elicited Ca2+ increases. D, bars indicate the percentage of the F340/F380 relative to an initial application of 10 µM 5-HT (solid bars) or 10 mM caffeine (open bars) in the presence of the agents listed. Dashed line represents the height of the Ca2+ response due to 10 µM 5-HT before FLA 365 application. Significant difference relative to the initial 5-HT stimulation denoted by *, P < 0.05 and to 10 mM caffeine stimulation in the absence of FLA 365. ***, P < 0.01 by a paired t test. Number in parentheses is the number of cells examined. Error bars represent ± S.E.M.

Figure 7D summarizes data showing the percentage of change in the peak height of the F₃₄₀/F₃₈₀ response when comparisons are made between cells exposed twice to 10 µM 5-HT in the absence of antagonists (i.e., control; Fig. 7A) or absence and then presence of 2 or 20 µM FLA 365. The figure illustrates that 20 µM but not 2 µM FLA 365 reduces the amplitude of the Ca²⁺ responses due to 10 µM 5-HT in canine PASMCs. The Ca²⁺ response to 10 µM 5-HT was 91 ± 5% of the initial 5-HT response in cells that were not treated with any antagonists, 104 ± 5% in presence of 2 µM FLA 365, and 76 ± 9% in the presence of 20 µM FLA 365. Figure 7D also shows comparative effects on caffeine induced Ca²⁺ responses in those cells treated with 2 µM FLA 365, where the Ca²⁺ increase to 10 mM caffeine was substantially reduced, being 25 ± 4% of control.

The potential for complex interactions between InsP₃ and RyR Ca²⁺ signaling in vascular myocytes has the potential to lead to misinterpretation of the preceding findings. Therefore, a series of experiments to verify that FLA 365 can block InsP₃ receptor-mediated responses was conducted in HEK-293 cells, which express predominantly InsP₃ receptors. Although RyRs may be expressed in HEK-293 cells in early passages (Luo et al., 2005), the cells examined did not exhibit Ca²⁺ responses to 10 mM caffeine (data not shown). To further limit possible RyR contamination, caffeine was applied in every experiment to ensure that cells expressed InsP₃ receptors but not RyRs. Figure 8, A and B, show representative traces of InsP₃ receptor-mediated responses elicited with 100 µM CCh in the absence and presence of 2 or 20 µM FLA 365. Figure 8C summarizes the data showing 20 µM FLA 365 inhibited CCh-mediated Ca²⁺ elevations by 52 ± 1%. Yet, 2 µM FLA 365, which substantially inhibits RyR-mediated Ca²⁺ responses in PASMCs (Figs. 1 and 7), did not reduce Ca²⁺ responses to CCh (107 ± 3% of control). Figure 8D illustrates that washing cells exposed to 20 µM FLA 365 allowed for full recovery of the Ca²⁺ response to CCh, being 99 ± 4% of the control response. Thus, increasing the FLA 365 concentration by an order of magnitude above that needed to reduce RyR activity reversibly inhibits InsP₃ receptor activation.

View larger version (17K): [in this window] [in a new window]   Fig. 8. FLA 365 blocks CCh-elicited Ca2+ responses in HEK-293 cells. A, 2 µM FLA effects on 100 µM CCh-induced Ca2+ increases. B, 20 µM FLA effects on 100 µM CCh-elicited Ca2+ increases. C, bars indicate the percentage of the F340/F380 for 100 µM CCh in the absence relative to the presence of the listed agents. D, bars show the change in cytosolic [Ca2+] from resting due 100 µM CCh in the absence, presence, and following washout of 20 µM FLA 365. Dashed line represents the magnitude of the Ca2+ response due to 100 µM CCh before FLA 365 application. ***, significant difference relative to control by repeated measures ANOVA with a Newman-Keuls multiple comparison test (P < 0.001). , significant difference between control and 20 µM FLA 365 group by one-way ANOVA with a Newman-Keuls multiple comparison test (P < 0.001). Number in parentheses is the number of cells examined. Error bars represent ± S.E.M.

	Discussion

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FLA 365 has a number of characteristics that are significant to its general utility. FLA 365 inhibition of RyR is readily reversible, and exhibits some selectivity for RyR over inhibition of Ca_V and InsP₃ receptors. Low micromolar FLA 365 concentrations significantly reduce, but do not eliminate, RyR activation without effect on InsP₃-related responses. FLA 365 at high concentrations blocks Ca_V in canine PASMCs, and 5-HT- and CCh-generated Ca²⁺ release in either canine PASMCs or HEK-293 cells. Although this latter effect of FLA 365 on receptor-mediated Ca²⁺ responses is presumed to be through inhibition of InsP₃ receptors, the experimental design does not delineate whether FLA 365 may alter ligand activation of 5-HT or muscarinic receptors or generation of InsP₃ by phospholipase C. The differences in potency toward RyR compared with Ca_V- and InsP₃-generated Ca²⁺ responses indicates this compound may have untoward actions on other Ca²⁺-permeable channels. What is more, the data suggest that FLA 365 may inhibit InsP₃ responses more effectively in HEK-293 cells than in canine pulmonary arterial myocytes. The IC₅₀ for RyR inhibition in canine PASMCs is also somewhat dissimilar from that in skeletal and cardiac muscle (Chiesi et al., 1988; Calviello and Chiesi, 1989; Mack et al., 1992). These findings show that attention should be given to the selectivity as well as potency when FLA 365 is used to study RyR function.

The pharmacological properties of FLA 365 differ from other RyR antagonists and agents used to deplete Ca²⁺ stored in the SR. Ryanodine is time-, dose-, and use-dependent, which limits its utility in live-cell studies. This is evidenced by depression of InsP₃R-mediated responses due to leak of Ca²⁺ from the SR through RyRs locked in an open subconductance state. Tetracaine and dantrolene are similar to FLA 365 in that they too may block InsP₃ responses independently of the involvement of RyRs, whereas neomycin shares inhibition of Ca_V channels (Canzoniero et al., 1993; Lin et al., 1993; Fellner and Arendshorst, 2005; Wang et al., 2005). Cyclopiazonic acid and thapsigargin are two routinely used SERCA inhibitors that passively deplete the SR Ca²⁺ stores (Janiak et al., 2001; Wilson et al., 2002). This passive depletion can activate capacitative Ca²⁺ entry and reduce both InsP₃- and RyR-related Ca²⁺ responses (Janiak et al., 2001; Wilson et al., 2002). Therefore, sarcoplasmic reticulum Ca²⁺ store depletion would confound selective examination of InsP₃R or RyR activity as well as studies regarding the coupling between Ca²⁺-permeable channels.

Coupling of RyRs to InsP₃ receptors is important to smooth muscle function; yet, the extent of these interactions and their functions are poorly understood. Angiotensin II activates both InsP₃ as well as RyR pathways in rat renal arteries (Fellner and Arendshorst, 2005), and RyRs are important during norepinephrine-induced contractility and Ca²⁺ signaling in rat pulmonary arteries and myocytes (Zheng et al., 2005). Zheng et al. (2005) used a variety of RyR antagonists, including dantrolene, tetracaine, RuR, and ryanodine to evaluate the role of RyR activity during norepinephrine-induced Ca²⁺ responses and arterial contractility. FLA 365 would be useful in studies such as these because it is readily reversible and exhibits some selectivity for RyRs over InsP₃ receptors.

The experiments presented here do not provide any conclusive evidence for or against possible interactions between RyR and InsP₃R in canine PASMCs. However, our previous work suggests there is little direct activation of RyR during InsP₃R activity in these cells, because the RyR Ca²⁺ stores can be depleted without impacting angiotensin II induced, InsP₃-related Ca²⁺ release (Janiak et al., 2001). However, there may be species-related differences, because Zheng et al. (2005) provide evidence for interactions between InsP₃-related and RyR-mediated Ca²⁺ responses and functionality in rat pulmonary arteries and myocytes. The functional organization of the RyR and InsP₃ Ca²⁺ release pathways would likely be important to arterial reactivity during neurohumoral stimulation, and FLA 365 may be a useful reagent when assessing the organization of these pathways.

The coupling of Ca_V stimulation to RyR activation has also been evaluated in smooth muscle with RyRs underlying Ca²⁺ spark events evoked in response to membrane depolarization and Ca_V activation (Collier et al., 2000). Ryanodine was used in these real-time laser scanning confocal microscopy experiments to demonstrate the role of RyR to the Ca²⁺ spark events; FLA 365 would have advantages in these types of studies, because it is rapidly acting and reversible. In particular, our studies indicate low concentrations of FLA 365 would reduce RyR activity, allowing for spatial and temporal examinations of RyR coupling to other Ca²⁺-permeable channels, such as Ca_V and InsP₃ receptors. Secondarily, the preparation should recover following compound washout allowing for additional evaluations in the same preparation.

The mechanism of FLA 365 inhibition of the RyR is important to its usefulness. In particular, kinetic Ca²⁺ uptake and release studies performed on skeletal and cardiac SR vesicles showed that FLA 365 inhibited Ca²⁺ release monophasically, with an IC₅₀ of 3.4 µM (Calviello and Chiesi, 1989). The action of FLA 365 was synergistic with neomycin and ruthenium red in skeletal and cardiac SR vesicles (Chiesi et al., 1988; Calviello and Chiesi, 1989). Mack et al. (1992) provided evidence of multiple binding sites for FLA 365 in the same preparation, where FLA 365 could compete for occupation of one of the RyR binding sites, whereas neomycin or ruthenium red would occupy the other (Mack et al., 1992). Our data suggest that as many as two FLA 365 molecules may be required to inhibit the RyR in canine PASMCs, signifying that the actions of FLA 365 may be complex.

FLA 365 is an important RyR inhibitor in that it does not allow for long-lived RyR channel openings, which contrasts the effects of ryanodine. It has previously been suggested that FLA 365 may act as an open channel inhibitor, where the FLA may bind when the RyR is activated, and there is maximal Ca²⁺ efflux rate. Yet, FLA 365 does not induce the subconductance states that underlie the long-lived RyR channel openings (Calviello and Chiesi, 1989; Mack et al., 1992). Our data suggest that FLA 365 is not likely to cause these long-lived channel openings in smooth muscle myocytes, because short incubations were sufficient to reduce caffeine elicited Ca²⁺ responses. Moreover, the slow Ca²⁺ rise generally afforded with ryanodine (e.g., Fig. 2B) was not observed in most cells.

FLA 365 concentrations >20 µM induced small-amplitude Ca²⁺ transients that may be due to short-lived RyR channel openings that occur before FLA 365 can bind fully and completely block channel activity. Even still, this phenomenon was not observed at 2 µM FLA 365, concentrations that would be more typically used to antagonize RyR activity.

The data presented provide evidence that Ca_V does not contribute significantly to the peak height of Ca²⁺ release due to caffeine or 5-HT in canine pulmonary myocytes. Our recently published work indicates that 5-HT-elicited Ca²⁺ increases were not markedly affected when Ca_V was inhibited (Wilson et al., 2005). In this same report, Ca_V is shown to be important to sustained 5-HT-mediated Ca²⁺ responses and arterial contractility, which is common among other G protein-coupled pathways in arteries (Wilson et al., 2005). However, the present studies do provide further support for the premise that transient Ca²⁺ responses due to RyR or InsP₃ receptor activation do not significantly recruit Ca_V Ca²⁺ entry pathways in canine PASMCs.

Overall, this study offers evidence that FLA 365 inhibits RyRs in smooth muscle. Albeit, further investigations could be performed to assess potential limitations due to inhibition of other mechanisms of Ca²⁺ signaling, such as the mitochondrial uniporter, which may be a RyR (Beutner et al., 2001, 2005) or nonselective cation channels. Even though FLA 365 has a narrow selectivity window and it does not fully inhibit RyRs, its rapid reversibility and distinctive properties may provide researchers with a useful pharmacological tool to examine intracellular Ca²⁺ signaling dynamics in vascular smooth muscle and other preparations.

	Acknowledgements

 
We thank Matthew Loftin, Will Graugnard, Phillip Keller, and Shen Xiao-Ming for technical assistance.

	Footnotes

 
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.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.122119.

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; Ca_v, 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; I_Ba, 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.

¹ Current affiliation: Department of Pharmacology University of Tennessee, Memphis, Tennessee.

Address correspondence to: Dr. Sean M. Wilson, University of Mississippi School of Pharmacy, University of Mississippi, 303 Faser Hall, University, MS 38677. E-mail: wilson{at}olemiss.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Ask AL, Fagervall I, Florvall L, Ross SB, and Ytterborn S (1985) Inhibition of monoamine oxidase in 5-hydroxytryptaminergic neurones by substituted p-aminophenylalkylamines. Br J Pharmacol 85: 683–690. 3861206

Ask AL and Ross SB (1987) Inhibition of 5-hydroxytryptamine accumulation and deamination by substituted phenylalkylamines in hypothalamic synaptosomes from normal and reserpine-pretreated rats. Naunyn Schmiedebergs Arch Pharmacol 336: 591–596. 2965308[CrossRef][Medline]

Baylor SM and Hollingworth S (2000) Measurement and interpretation of cytoplasmic [Ca²⁺] signals from calcium-indicator dyes. News Physiol Sci 15: 19–26. 11390871[Abstract/Free Full Text]

Beutner G, Sharma VK, Giovannucci DR, Yule DI, and Sheu SS (2001) Identification of a ryanodine receptor in rat heart mitochondria. J Biol Chem 276: 21482–21488. 11297554[Abstract/Free Full Text]

Beutner G, Sharma VK, Lin L, Ryu SY, Dirksen RT, and Sheu SS (2005) Type 1 ryanodine receptor in cardiac mitochondria: transducer of excitation-metabolism coupling. Biochim Biophys Acta 1717: 1–10. 16246297[Medline]

Bleakman D, Brorson JR, and Miller RJ (1990) The effect of capsaicin on voltagegated calcium currents and calcium signals in cultured dorsal root ganglion cells. Br J Pharmacol 101: 423–431. 1701680[Medline]

Calviello G and Chiesi M (1989) Rapid kinetic analysis of the calcium-release channels of skeletal muscle sarcoplasmic reticulum: the effect of inhibitors. Biochemistry 28: 1301–1306. 2469467[CrossRef][Medline]

Canzoniero LM, Taglialatela M, Di Renzo G, and Annunziato L (1993) Gadolinium and neomycin block voltage-sensitive Ca²⁺ channels without interfering with the Na(+)-Ca²⁺ antiporter in brain nerve endings. Eur J Pharmacol 245: 97–103. 8491259[CrossRef][Medline]

Catterall WA, Perez-Reyes E, Snutch TP, and Striessnig J (2005) International Union of Pharmacology. XLVIII. Nomenclature and structure-function relationships of voltage-gated calcium channels. Pharmacol Rev 57: 411–425. 16382099[Abstract/Free Full Text]

Charuk JH, Pirraglia CA, and Reithmeier RA (1990) Interaction of ruthenium red with Ca2(+)-binding proteins. Anal Biochem 188: 123–131. 1699445[CrossRef][Medline]

Chen SR and MacLennan DH (1994) Identification of calmodulin-, Ca(2+)-, and ruthenium red-binding domains in the Ca²⁺ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum. J Biol Chem 269: 22698–22704. 7521330[Abstract/Free Full Text]

Chiesi M, Schwaller R, and Calviello G (1988) Inhibition of rapid Ca-release from isolated skeletal and cardiac sarcoplasmic reticulum (SR) membranes. Biochem Biophys Res Commun 154: 1–8. 2456059[CrossRef][Medline]

Cibulsky SM and Sather WA (1999) Block by ruthenium red of cloned neuronal voltage-gated calcium channels. J Pharmacol Exp Ther 289: 1447–1453. 10336538[Abstract/Free Full Text]

Collier ML, Ji G, Wang Y, and Kotlikoff MI (2000) Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release. J Gen Physiol 115: 653–662. 10779321[Abstract/Free Full Text]

del Corsso C, Ostrovskaya O, McAllister CE, Murray K, Hatton WJ, Gurney AM, Spencer NJ, and Wilson SM (2006) Effects of aging on Ca2+ signaling in murine mesenteric arterial smooth muscle cells. Mech Ageing Dev 127: 315–323. 16413046[CrossRef][Medline]

Dray A, Forbes CA, and Burgess GM (1990) Ruthenium red blocks the capsaicin-induced increase in intracellular calcium and activation of membrane currents in sensory neurones as well as the activation of peripheral nociceptors in vitro. Neurosci Lett 110: 52–59. 1691472[CrossRef][Medline]

Fellner SK and Arendshorst WJ (2005) Angiotensin II Ca²⁺ signaling in rat afferent arterioles: stimulation of cyclic ADP ribose and IP3 pathways. Am J Physiol Renal Physiol 288: F785–F791. 15598842[Abstract/Free Full Text]

Grynkiewicz G, Poenie M, and Tsien RY (1985) A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450. 3838314[Abstract/Free Full Text]

Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391: 85–100. 6270629[CrossRef][Medline]

Hirano M, Imaizumi Y, Muraki K, Yamada A, and Watanabe M (1998) Effects of ruthenium red on membrane ionic currents in urinary bladder smooth muscle cells of the guinea-pig. Pflugers Arch 435: 645–653. 9479017[CrossRef][Medline]

Jaggar JH, Wellman GC, Heppner TJ, Porter VA, Perez GJ, Gollasch M, Kleppisch T, Rubart M, Stevenson AS, Lederer WJ, et al. (1998) Ca²⁺ channels, ryanodine receptors and Ca(2+)-activated K+ channels: a functional unit for regulating arterial tone. Acta Physiol Scand 164: 577–587.[CrossRef][Medline]

Janiak R, Wilson SM, Montague S, and Hume JR (2001) Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells. Am J Physiol Cell Physiol 280: C22–C33. 11121373[Abstract/Free Full Text]

Janssen LJ and Sims SM (1992) Acetylcholine activates non-selective cation and chloride conductances in canine and guinea-pig tracheal myocytes. J Physiol 453: 197–218. 1281502[Abstract/Free Full Text]

Lin MJ and Lin-Shiau SY (1996) Ruthenium red, a novel enhancer of K+ currents at mouse motor nerve terminals. Neuropharmacology 35: 615–623. 8887970[CrossRef][Medline]

Lin X, Hume RI, and Nuttall AL (1993) Voltage-dependent block by neomycin of the ATP-induced whole cell current of guinea-pig outer hair cells. J Neurophysiol 70: 1593–1605. 7506758[Abstract/Free Full Text]

Luo D, Sun H, Xiao RP, and Han Q (2005) Caffeine induced Ca²⁺ release and capacitative Ca²⁺ entry in human embryonic kidney (HEK293) cells. Eur J Pharmacol 509: 109–115. 15733545[CrossRef][Medline]

Ma J (1993) Block by ruthenium red of the ryanodine-activated calcium release channel of skeletal muscle. J Gen Physiol 102: 1031–1056. 7510773[Abstract/Free Full Text]

Mack WM, Zimanyi I, and Pessah IN (1992) Discrimination of multiple binding sites for antagonists of the calcium release channel complex of skeletal and cardiac sarcoplasmic reticulum. J Pharmacol Exp Ther 262: 1028–1037. 1382127[Abstract/Free Full Text]

MacMillan D, Chalmers S, Muir TC, and McCarron JG (2005) IP3-mediated Ca2+ increases do not involve the ryanodine receptor, but ryanodine receptor antagonists reduce IP3-mediated Ca2+ increases in guinea-pig colonic smooth muscle cells. J Physiol 569: 533–544. 16195318[Abstract/Free Full Text]

Nagata K, Duggan A, Kumar G, and Garcia-Anoveros J (2005) Nociceptor and hair cell transducer properties of TRPA1, a channel for pain and hearing. J Neurosci 25: 4052–4061. 15843607[Abstract/Free Full Text]

Pessah IN and Zimanyi I (1991) Characterization of multiple [³H]ryanodine binding sites on the Ca²⁺ release channel of sarcoplasmic reticulum from skeletal and cardiac muscle: evidence for a sequential mechanism in ryanodine action. Mol Pharmacol 39: 679–689. 1851961[Abstract]

Rossi CS, Vasington FD, and Carafoli E (1973) The effect of ruthenium red on the uptake and release of Ca²⁺ by mitochondria. Biochem Biophys Res Commun 50: 846–852. 4689082[CrossRef][Medline]

Smith JS, Imagawa T, Ma J, Fill M, Campbell KP, and Coronado R (1988) Purified ryanodine receptor from rabbit skeletal muscle is the calcium-release channel of sarcoplasmic reticulum. J Gen Physiol 92: 1–26. 2459298[Abstract/Free Full Text]

Wang C, Du XN, Jia QZ, and Zhang HL (2005) Binding of PLCdelta1PH-GFP to PtdIns(4,5)P2 prevents inhibition of phospholipase C-mediated hydrolysis of PtdIns(4,5)P2 by neomycin. Acta Pharmacol Sin 26: 1485–1491. 16297348[CrossRef][Medline]

Wann KT and Richards CD (1994) Properties of single calcium-activated potassium channels of large conductance in rat hippocampal neurons in culture. Eur J Neurosci 6: 607–617. 7517771[CrossRef][Medline]

Wilson SM, Mason HS, Ng LC, Montague S, Johnston L, Nicholson N, Mansfield S, and Hume JR (2005) Role of basal extracellular Ca2+ entry during 5-HT-induced vasoconstriction of canine pulmonary arteries. Br J Pharmacol 144: 252–264. 15655514[CrossRef][Medline]

Wilson SM, Mason HS, Smith GD, Nicholson N, Johnston L, Janiak R, and Hume JR (2002) Comparative capacitative calcium entry mechanisms in canine pulmonary and renal arterial smooth muscle cells. J Physiol 543: 917–931. 12231648[Abstract/Free Full Text]

Zheng YM, Wang QS, Rathore R, Zhang WH, Mazurkiewicz JE, Sorrentino V, Singer HA, Kotlikoff MI, and Wang YX (2005) Type-3 ryanodine receptors mediate hypoxia-, but not neurotransmitter-induced calcium release and contraction in pulmonary artery smooth muscle cells. J Gen Physiol 125: 427–440. 15795312[Abstract/Free Full Text]

Zhuge R, Fogarty KE, Tuft RA, and Walsh JV Jr (2002) Spontaneous transient outward currents arise from microdomains where BK channels are exposed to a mean Ca²⁺ concentration on the order of 10 microM during a Ca(2+) spark. J Gen Physiol 120: 15–27. 12084772[Abstract/Free Full Text]

ZhuGe R, Sims SM, Tuft RA, Fogarty KE and Walsh JV, Jr. (1998) Ca2+ sparks activate K+ and Cl^– channels, resulting in spontaneous transient currents in guinea-pig tracheal myocytes. J Physiol 513: 711–718. 9824712[Abstract/Free Full Text]

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