Submaximal stimulation of porcine endothelial cells causes focal Ca2+ elevation beneath the cell membrane
Authors: Graier Wolfgang F. , Paltauf‐Doburzynska Jolanta , Hill Brent J. F. , Fleischhacker Eleonore , Hoebel Bernhard G. , Kostner Gert M. , Sturek Michael 
Published: September 2004

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).

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.

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 Ca2+ entry pathways but stimulation via intracellular Ca2+ release.

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 μmol l−1 ryanodine, indicating the involvement of Ca2+‐induced Ca2+ release (CICR).

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.

Treatment with 10 μmol l−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.

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.

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.