Stealth ryanodine-sensitive Ca2+ release contributes to activity of capacitative Ca2+ entry and nitric oxide synthase in bovine endothelial cells

Abstract

1. The involvement of ryanodine-sensitive Ca2+ release (RsCR) in bradykinin (Bk)-induced Ca2+ release, capacitative Ca2+ entry (CCE) and nitric oxide synthase (NOS) activation was assessed in freshly isolated bovine coronary artery endothelial cells. 2. Using deconvolution microscopy fura-2 was found throughout the whole cytosol, while the cell membrane impermeable dye FFP-18 was exclusively in the cell membrane. Thus, perinuclear ([Ca2+]pn) and subplasmalemmal Ca2+ concentration ([Ca2+]sp) were monitored using fura-2 and FFP-18. 3. Inhibition of Na+-Ca2+ exchange by lowering extracellular Na+ concentration augmented the Bk-induced [Ca2+]pn signal in Ca2+-free solution. This effect was abolished when RsCR was prevented with 25 micromol l-1 ryanodine, while inhibition of RsCR had no effect on Bk-induced increase in [Ca2+]pn without inhibition of Na+-Ca2+ exchange. 4. Initiating RsCR by 200 nmol l-1 ryanodine increased [Ca2+]sp, while [Ca2+]pn remained constant. However, when Na+-Ca2+ exchange was prevented, ryanodine was also able to elevate [Ca2+]pn. 5. Blockage of RsCR diminished Ca2+ extrusion in response to stimulation with Bk in normal Na+-containing solution. 6. Inhibition of RsCR blunted Bk-activated CCE, while inhibition of Na+-Ca2+ exchange during stimulation enhanced CCE. 7. Although direct activation of RsCR failed to activate NOS, inhibition of RsCR diminished the effect of ATP and Bk on NOS, while the effect of thapsigargin remained unchanged. 8. These data suggest that during stimulation subplasmalemmal RsCR occurs, which contributes to the activities of CCE and NOS. Thus, the function of the subplasmalemmal Ca2+ control unit must be extended as a regulator for CCE and NOS.

Figure 1. Effect of low extracellular [Na⁺]_o on agonist-induced changes of [Ca²⁺]_pn in endothelial cells freshly isolated from bovine left circumflex arteries

Freshly isolated endothelial cells were loaded with fura-2 to measure [Ca²⁺]_pn. In Ca²⁺-free HBS containing 145 (145 [Na⁺]_o, ○, continuous line) or 19 (19 [Na⁺]_o, •, dashed line) mmol l⁻¹ Na⁺, cells were stimulated for 1 min with 100 nmol l⁻¹ Bk followed by an addition of 2.5 mmol l⁻¹ Ca²⁺ as indicated. In experiments using 19 mmol l⁻¹ Na⁺-containing solution, [Na⁺]_o was changed to 145 mmol l⁻¹ at time 3 min. Points represent means ±s.e.m. (n= 22).

Figure 2. Effect of 25 μmmu;mol l⁻¹ ryanodine on Bk-induced changes of [Ca²⁺]_pn in single freshly isolated bovine left circumflex endothelial cells in 145 (panel A) and 19 (panel B) mmol l⁻¹ Na⁺-containing solution

Freshly isolated endothelial cells were loaded with fura-2 to measure [Ca²⁺]_pn. Panel A, in Ca²⁺-free HBS containing 145 mmol l⁻¹ Na⁺ without (control, ○, continuous line) or with 25 μmmu;mol l⁻¹ ryanodine (•, dashed line) endothelial cells were stimulated with 100 nmol l⁻¹ Bk followed by the addition of 2.5 mmol l⁻¹ Ca²⁺ as indicated. Panel B, in Ca²⁺-free HBS containing 19 mmol l⁻¹ Na⁺ without (control, ○, continuous line) or with 25 μmmu;mol l⁻¹ ryanodine (•, dashed line) cells were stimulated with 100 nmol l⁻¹ Bk followed by the addition of 2.5 mmol l⁻¹ Ca²⁺. As indicated, [Na⁺]_o was increased to 145 mmol l⁻¹. Points represent means ±s.e.m. (panel A, n= 36; panel B, n= 32).

Figure 3

Summary of the results on the effect of Bk on intracellular Ca²⁺ release (A) and CCE (B) in normal and low Na⁺-containing solution in the absence and presence of ryanodine.

Figure 4. Prevention of Na⁺-Ca²⁺ exchange by lowering [Na⁺]_o unmasked ryanodine-induced Ca²⁺ release

Single endothelial cells were loaded with fura-2 to measure [Ca²⁺]_pn. As indicated, cells were stimulated with 200 nmol l⁻¹ ryanodine (RY) in Ca²⁺-free HBS containing 145 (RY, ○, continuous line) or 19 mmol l⁻¹ Na⁺ (RY, •, dashed line). Each point represents the mean ±s.e.m. (n= 24).

Figure 5. Distribution of FFP-18 and fura-2 in the bovine left circumflex coronary artery endothelial cells and in EA.hy 926 cells

Freshly isolated bovine left circumflex coronary artery endothelial cells (panels A and B) and cultured EA.hy 926 (panels C and D) were loaded with FFP-18 (panels A and C) or fura-2 free acid (panels B and D) using laser-stress wave loading technique (Graier et al. 1998), equilibrated for 10 min at room temperature, washed three times in HBS and placed in a glass-bottomed chamber. Fluorescence images were collected using a × 100 objective (NA 1.3) with an excitation of 340 ± 5 nm and emission of 510 ± 10 nm for both dyes (5 to 10 s exposure time). Panels A and B: 2-D image of a slice in the middle depth of freshly isolated bovine endothelial cells. Panels C and D: 3-D reconstructed image of EA.hy 926 cells. Images were collected throughout the whole cell with 0.3 μmmu;m interslice distance, and out-of-focus fluorescence was removed using the advanced constrained iterative algorithm (MicroTome, Vaytek, Turnbridge Wells, UK). 3-D reconstruction was performed with VoxBlast (Vaytek, Turnbridge Wells, UK). In the case of FFP-18 (panel C) just 10 slices at the cell equator are shown, while panel D (fura-2) shows the whole cell. Identical distribution of fura-2 was found when fura-2 was loaded conventionally using 2 μmmu;mol l⁻¹ fura-2 AM over 30 min.

Figure 6. Ryanodine induced subplasmalemmal Ca²⁺ increase in bovine coronary endothelial cells monitored by FFP-18

Endothelial cells freshly isolated from bovine circumflex coronary artery were loaded with FFP-18 using laser-generated stress waves to monitor changes in [Ca²⁺]_sp. After 10 min equilibration time at room temperature, cells were washed twice, resuspended in Ca²⁺-free HBS and placed into a stirred cuvette. Changes in [Ca²⁺]_sp were monitored at 335 and 365 nm excitation and 500 nm emission. Each point represents the mean ±s.e.m. (n= 14).

Figure 7. Inhibition of ryanodine-sensitive Ca²⁺ release attenuates Ca²⁺ extrusion in response to bradykinin

Suspended cultured endothelial cells (passage 2; 7 × 10⁷ cells ml⁻¹) were stimulated with 100 nmol l⁻¹ Bk in the absence (control, ○, continuous line) or presence (•, dashed line) of 25 μmmu;mol l⁻¹ ryanodine in Ca²⁺-free HBS containing 1 μmmu;mol l⁻¹ fura-2 dextran. The Ca²⁺ extrusion was monitored at 340 and 364 nm excitation and 500 nm emission. Each point represents the mean ±s.e.m. (n= 6).

Figure 8. Correlation of Ca²⁺ release and CCE on repetitive stimulation with Bk in normal and low [Na⁺] conditions in the absence and presence of ryanodine

Panel A shows the experimental protocol: fura-2-loaded endothelial cells freshly isolated from bovine circumflex arteries were stimulated with 100 nmol l⁻¹ Bk for 2 min either at time point 1 min (1), at time points 1 and 4 min (2) or at time points 1, 4 and 7 min (3). At time 9 min [Na⁺]_o was set to 19 mmol l⁻¹ and 2.5 mmol l⁻¹ Ca²⁺ was added at time 10 min. Panel B, elevation of [Ca²⁺]_pn to Bk at time 1 min (1), or the total sum of the increases of [Ca²⁺]_pn upon two (2: 1st peak plus 2nd peak) or three (3: 1st peak plus 2nd peak plus 3rd peak) stimulations is shown on the x-axis. The increase in [Ca²⁺]_pn to the addition of Ca²⁺ is shown on the y-axis. Bk stimulation was performed in Ca²⁺-free HBS containing 145 mmol l⁻¹ Na⁺ (○), 19 mmol l⁻¹ Na⁺ (•), 145 mmol l⁻¹ Na⁺ and 25 μmmu;mol l⁻¹ ryanodine (□) and 19 mmol l⁻¹ Na⁺ plus 25 μmmu;mol l⁻¹ ryanodine (▪). Each point represents the mean ±s.e.m. (n= 12).

Figure 9. Effect of ryanodine-induced RsCR on NOS activity (A) and changes in autacoid-induced NOS activation by inhibition of RsCR with a high concentration of ryanodine (B)

Cultured endothelial cells from the bovine circumflex coronary artery (passage 2) were stimulated with 20, 200 and 500 nmol l⁻¹ ryanodine (panel A), or with ATP (10 and 100 μmmu;mol l⁻¹), Bk (10 and 100 nmol l⁻¹) and 2 μmmu;mol l⁻¹ thapsigargin (TG) in the absence or presence of 5 μmmu;mol l⁻¹ ryanodine (panel B) for 15 min in HBS. Columns represent the percentage deviation from basal NOS activity (panel A) or the percentage of conversion of [³H]-L-arginine to [³H]-l-citrulline (panel B). Each column represents the mean ±s.e.m. (A, n= 4; B, n= 5). *P < 0.05 vs. absence of ryanodine.