Submaximal stimulation of porcine endothelial cells causes focal Ca2+ elevation beneath the cell membrane

Abstract

1. Endothelial cell activation is correlated with increased cytosolic Ca2+ concentration, often monitored with cytoplasmic Ca2+ dyes, such as fura-2 and Calcium Green-1. We tested the hypothesis that during weak stimulation of porcine coronary artery endothelial cells, focal, subplasmalemmal Ca2+ elevations occur which are controlled by cell membrane Na(+)-Ca2+ exchange near mitochondrial membrane and superficial endoplasmic reticulum (SER). 2. Bulk Ca2+ concentration ([Ca2+]b) was monitored using fura-2 or Calcium Green-1 and subplasmalemmal Ca2+ concentration ([Ca2+]sp) was determined with FFP-18. The distribution of the SER network was estimated using laser scanning and deconvolution microscopy. 3. Sodium fluoride (10 mmol l-1) and submaximal concentrations of bradykinin (Bk; 1 nmol l-1) stimulated Ca2+ entry with no increase in [Ca2+]b. Although inositol 1,4,5-trisphosphate formation and intracellular Ca2+ release in response to both stimuli were similar, Ca2+ entry in response to NaF exceeded that in response to 1 nmol l-1 BK by fourfold, suggesting additional effects of NaF on Ca+ entry pathways but stimulation via intracellular Ca2+ release. 4. Prevention of Na(+)-Ca2+ exchange activity by decreasing extracellular Na+ unmasked intracellular Ca2+ release in response to NaF and 1 nmol l-1 Bk, indicated by an increase in [Ca2+]b. Thereby, NaF depleted Bk-releasable Ca2+ pools, while mitochondrial Ca2+ content (released with FCCP or oligomycin) and the amount of Ca2+ stored within the cells (released with ionomycin) was increased compared with cells treated with NaF under normal Na+ conditions. The NaF-initiated increase in [Ca2+]b and depletion of Bk-releasable Ca2+ pool(s) in the low-Na+ condition was diminished by 25 mumol l-1 ryanodine, indicating the involvement of Ca(2+)-induced Ca2+ release (CICR). 5. In simultaneous recordings of [Ca2+]sp (with FFP-18) and [Ca2+]b (with Calcium Green-1), 1 nmol l-1 Bk or 10 mmol l-1 NaF yielded focal [Ca2+] elevation in the subplasmalemmal region with no increase in the perinuclear area. 6. Treatment with 10 mumol-1 nocodazole caused the SER to collapse and unmasked Ca2+ release in response to 1 nmol l-1 Bk and 10 mmol l-1 NaF, similar to low-Na+ conditions, while the effect of thapsigargin was not changed. 7. These data show that in endothelial cells, focal, subplasmalemmal Ca2+ elevations in response to small or slow IP3 formation occur due to vectorial Ca2+ release from the SER towards the plasmalemma followed by Ca2+ extrusion by Na(+)-Ca2+ exchange. While these local Ca2+ elevations are not detectable with Ca2+ dyes for the determination of [Ca2+]b, prevention of Ca2+ extrusion or SER disruption yields increases in [Ca2+]b partially due to CICR. 8. All of the data support our hypothesis that in weakly stimulated endothelial cells, intracellular Ca2+ release and [Ca2+] elevation are limited to the subplasmalemmal region. We propose that the SER co-operates with associated parts of the plasma membrane to control Ca2+ homeostasis, Ca2+ distribution and Ca2+ entry. The existence of such a subplasmalemmal Ca2+ control unit (SCCU) needs to be considered in discussions of Ca2+ signalling, especially when cytoplasmic Ca2+ dyes, such as fura-2 or Calcium Green-1, are used.

Figure 1. Effect of bradykinin and NaF on cultured porcine endothelial inositol 1,4,5-trisphosphate (IP₃) levels

A, comparison of the time course of IP₃ formation in response to NaF and bradykinin. Endothelial cells were preincubated with myo-[³H]inositol for 48 h in DMEM. In the nominal absence of extracellular Ca²⁺, cells were stimulated with either 100 nmol l⁻¹ bradykinin (^) or 10 mmol l⁻¹ NaF (•) and experiments were stopped at times indicated. Levels of endothelial IP₃ were expressed as percentage increases of the IP₃ levels in cells under resting conditions. B, comparison of the IP₃ formation within 30 s to 1, 10, 30, 100 and 1000 nmol l⁻¹ bradykinin (•) and 10 mmol l⁻¹ NaF (column) measured by radioactive binding assay. Each point represents the mean ±s.e.m. (n = 4-6).

Figure 2. Representative tracings of the intracellular Ca²⁺ signalling in cultured endothelial cells in monolayer stimulated with bradykinin (A) and NaF (B)

Agonists were added in nominal Ca²⁺-free solution followed by the addition of 2.5 mmol l⁻¹ Ca²⁺, indicated by the bars. A, endothelial Ca²⁺ response to 100 nmol l⁻¹ bradykinin was monitored with Calcium Green-1, and increased bulk Ca²⁺ ([Ca²⁺]_b) is demonstrated by changes in the fluorescence intensity (F.I.) of the Ca²⁺-sensitive wavelength of Calcium Green-1 measured at 506 nm excitation and 534 nm emission. B, effect of 10 mmol l⁻¹ NaF on intracellular Ca²⁺ in fura-2-loaded cells in the nominal absence of extracellular Ca²⁺ followed by an addition of 2.5 mmol l⁻¹ Ca²⁺ (^, continuous line). Addition of 2.5 mmol l⁻¹ Ca²⁺ without pre-stimulation with NaF is shown also (•). Points represent means ±s.e.m. (n = 24).

Figure 3. Lowering extracellular sodium concentration unmasked NaF- (A and B) and bradykinin-induced (C) intracellular Ca²⁺ release

Single endothelial cells loaded with fura-2 were stimulated with 10 mmol l⁻¹ NaF (A) at various concentrations of bradykinin (C) in the nominal absence of extracellular Ca²⁺ in buffer containing 145 mmol l⁻¹ (normal Na⁺, ^, continuous line) or 19 mmol l⁻¹ extracellular Na⁺ (low Na⁺, □, dashed line). In experiments where 19 mmol l⁻¹ extracellular Na⁺ buffer was used, extracellular Na⁺ was changed to 145 mmol l⁻¹ at time 6 min. Each point represents the mean ±s.e.m. (n = 19 in A and n = 8 in C). B, correlation of the extracellular Na⁺ concentration used in the experiments with the NaF-induced (10 mmol l⁻¹) increases in intracellular free Ca²⁺ monitored by fura-2 in the nominal absence of extracellular Ca²⁺. Each point represents the mean ±s.e.m. (n = 6-19).

Figure 4. Effect of NaF in depleting the bradykinin-sensitive Ca²⁺ pool in normal and low-sodium buffer (A) and in the absence and presence of ryanodine (B)

A, single endothelial cells were loaded with fura-2 and stimulated in the nominal absence of extracellular Ca²⁺ with 10 mmol l⁻¹ NaF followed by 100 nmol l⁻¹ bradykinin (Bk). Experiments were performed in solution containing 145 mmol l⁻¹ Na⁺ (normal Na, ^, continuous line) and 19 mmol l⁻¹ Na⁺ (low Na, □, dashed plot). In experiments in 19 mmol l⁻¹ Na⁺ buffer, extracellular Na⁺ was changed to 145 mmol l⁻¹ at time 6 min. Each point represents the mean ±s.e.m. (n = 16). B, cells were stimulated in 19 mmol l⁻¹ Na⁺ buffer in nominal Ca²⁺-free solution for 2 min with 10 mmol l⁻¹ NaF in the absence (Control, ^, dashed line) or presence of 25 μmol l⁻¹ ryanodine (Ryanodine, •, continuous line). After 2.5 min extracellular Na⁺ was changed to 145 mmol l⁻¹ and NaF and ryanodine were washed out, followed by the addition of 100 nmol l⁻¹ Bk for 2 min. Each point represents the mean ±s.e.m. (n = 4).

Figure 5. Effect of NaF on overall Ca²⁺ stored within the cells (A) and on mitochondrial Ca²⁺ (B) in normal and low-sodium buffer

A, single endothelial cells were loaded with fura-2 and stimulated in the nominal absence of extracellular Ca²⁺ with 10 mmol l⁻¹ NaF followed by 5 μmol l⁻¹ ionomycin as bars indicate. Experiment was performed in solution containing 145 mmol l⁻¹ extracellular Na⁺ (normal Na, ^, continuous line) and 19 mmol l⁻¹ extracellular Na⁺ (low Na, □, dashed line). In experiments where 19 mmol l⁻¹ extracellular Na⁺ buffer was used, extracellular Na⁺ was changed to 145 mmol l⁻¹ at time 6 min. Each point represents the mean ±s.e.m. (n = 10-12). B, in cells pre-stimulated in the nominal absence of extracellular Ca²⁺ with 10 mmol l⁻¹ NaF, mitochondrial Ca²⁺ was released using 10 μmol l⁻¹ FCCP or 10 μmol l⁻¹ oligomycin. Columns represent the means ±s.e.m. (n = 4-8). *P < 0.05 vs. experiments in the presence of 145 mmol l⁻¹ extracellular Na⁺.

Figure 6. Representative tracings of the Ca²⁺ signalling in response to stimulation with 100 nmol l⁻¹ bradykinin measured by fura-2 (A) and Calcium Green-1 (B) after laser-pulse wave loading procedure and the effect of sub-maximal concentration of bradykinin (1 nmol l⁻¹) on suspended porcine endothelial Ca²⁺ concentration detected by FFP-18 (C and D) and Calcium Green-1 (D)

Cultured endothelial cells were harvested by enzymatic digestion, centrifuged and resuspended in DMEM containing 2 μmol l⁻¹ fura-2 pentapotassium salt (A) or 5 μmol l⁻¹ Calcium Green-1 hexapotassium salt (B). Dye loading was performed using laser pulse wave loading procedure (LSWP) as described in Methods. Experiments were performed in nominal Ca²⁺-free saline. As bars indicate, 100 nmol l⁻¹ bradykinin and 2.5 mmol l⁻¹ Ca²⁺ were added. C, effect of laser-pulse wave loading procedure for FFP-18 loading in endothelial cells. Endothelial cells were preincubated for 45 min with 2 μmol l⁻¹ FFP-18 at room temperature in the dark (continuous line, ○) or were loaded using LSWP (dashed line, •). Experiments were performed in the nominal absence of extracellular Ca²⁺. At times indicated 1 nmol l⁻¹ bradykinin or 2.5 mmol l⁻¹ Ca²⁺ were added. Each point represents the mean ±s.e.m. (n = 5). D, simultaneous recordings of subplasmalemmal Ca²⁺ concentration detected by FFP-18 (dashed line, •) and mean cytoplasmic Ca²⁺ concentration measured with Calcium Green-1 (continuous line, ○). Each point represents the mean ±s.e.m. (n = 4).

Figure 7. Confocal image of ryanodine receptor distribution in living endothelial cells

Freshly isolated endothelial cells were loaded with 10⁻⁷ mol l⁻¹ BODIPY FL-X ryanodine for 10 min and images were collected using a confocal microscope. The figure shows a transmitted light image (A), the corresponding confocal image at approximately the middle depth of the cell (B) and a composite image of A and B (C). D shows a 3-dimensional reconstruction of 13 optical sections taken at 1 μm steps in the z-axis and the inset presents the single section in the x-y plane taken at the arrow. E, a 3-dimensional reconstruction above the x-y plane shown in B. The red circles and lines indicate corresponding areas in B and E. In F the plasmalemma is indicated by the outer red circle defined by the transmitted light image given in A and C. The inner red circle shows an erosion of 3 pixels from the plasmalemma towards the centre of the cell, which represents a distance of 0.6 μm from the plasmalemma.

Figure 8. Effect of a nocodazole treatment on 3-dimensional organization of the endoplasmic reticulum in porcine endothelial cells using BODIPY FL-X ryanodine

Using deconvolution microscopy images were collected from cultured endothelial cells (passage 1) treated for 16 h in the absence (A) or presence (C) of 10 μmol l⁻¹ nocodazole. The corresponding 1 μm thick x-y slice in middle depth after deconvolution is shown in B for image A and in D for image C.

Figure 9. Effect of disruption of superficial ER compartments on bulk Ca²⁺ signal in response to NaF and a low concentration of bradykinin

Endothelial cells pre-treated with or without nocodazole for 16 h and loaded with fura-2 AM were stimulated with NaF (10 mmol l⁻¹) (A) and a sub-maximal concentration of bradykinin (i.e. 1 nmol l⁻¹; B). Cultured endothelial cells were incubated for 16 h in DMEM in the absence (continuous line, ^) or presence (dashed linel, •) of 10 μmol l⁻¹ nocodazole. Before the experiments, cells were washed three times and loaded with fura-2 AM for 45 min at room temperature in the dark. Experiments were performed in the absence of nocodazole in nominal Ca²⁺-free buffer. Points represent the means ±s.e.m. (n = 22).

Figure 10. Effect of a pre-treatment of cultured porcine endothelial cells with nocodazole on bulk Ca²⁺ concentration in cells stimulated with maximal concentration of bradykinin (A) and thapsigargin (B)

Cultured endothelial cells were incubated for 16 h in DMEM in the absence (continuous line, ^) or presence (dashed line, •) of 10 μmol l⁻¹ nocodazole. Before experiments, cells were washed three times and endothelial cells were stimulated in the absence of nocodazole with bradykinin (i.e. 100 nmol l⁻¹; A), and the ER ATPase inhibitor thapsigargin (1 μmol l⁻¹; B). Points represent the means ±s.e.m. (n = 6-9).

Figure 11. Schematic view of the proposed focal, subplasmalemmal Ca²⁺ elevation by weak stimulation of vascular endothelial cells

Small or slow formation of IP₃ by 1 nmol l⁻¹ bradykinin or 10 mmol l⁻¹ NaF initiates vectorial Ca²⁺ release from the subplasmalemmal endoplasmic reticulum (SER) towards the cell membrane. Under physiological conditions, Ca²⁺ release by the SER does not diffuse to deeper cytosolic compartments due to plasmalemmal Na⁺-Ca²⁺ exchange activity (i.e. subplasmalemmal Ca²⁺ control unit; SCCU). Thus, no bulk Ca²⁺ increase is observed. Prevention of plasmalemmal Na⁺-Ca²⁺ exchange by low extracellular Na⁺, allows subplasmalemmal released Ca²⁺ to diffuse into deeper parts of the cytosol, initiating Ca²⁺-induced Ca²⁺ release on the ryanodine receptors, resulting in depletion of IP₃-sensitive Ca²⁺ pool(s) and elevation of bulk Ca²⁺.