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

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          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.

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          Most cited references26

          • Record: found
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          Localization of endoplasmic reticulum in living and glutaraldehyde-fixed cells with fluorescent dyes.

          Certain fluorescent dyes, previously reported to localize mitochondria, when used at higher concentrations also localize a continuous net-like structure in both living and glutaraldehyde-fixed cells. A similar reticular structure can be detected by phase-contrast microscopy and whole-mount electron microscopy in potassium permanganate-fixed cells as well. This structure is mostly tubular, with some patch-like areas, and is likely to be the endoplasmic reticulum (ER). The organization of the reticular structure is sensitive to colchicine and rotenone but not to cytochalasin B, taxol, monensin, the calcium ionophore A23187, 12-O-tetradecanoylphorbol 13-acetate, or hydrocortisone.
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            A role for calcium influx in the regulation of mitochondrial calcium in endothelial cells.

            By using an endothelial cell line (ECV304), derived from human umbilical vein and transfected with recombinant aequorin targeted to the mitochondrial matrix, we find that stimulation with ATP evokes long lasting increases in mitochondrial Ca2+ ([Ca2+]m) that largely depend on Ca2+ influx. In these cells, the release of stored Ca2+ is inefficient at elevating [Ca2+]m. Consequently it appears that in ECV304 cells, bulk cytosolic Ca2+ ([Ca2+]c) is the main determinant of [Ca2+]m changes. In ECV304 cells < 4% of mitochondria are within 700 nm of the endoplasmic reticulum as opposed to 65% in HeLa cells, whereas 14% are within 700 nm of the inner surface of the plasma membrane, compared with < 6% in HeLa cells. Following Ca2+ depletion, readdition of extracellular Ca2+ evokes an increase in [Ca2+]m but not in [Ca2+]c. Under these conditions, microdomains of high [Ca2+]c may occur beneath the plasma membrane of ECV304 cells resulting in the preferential elevation of Ca2+ in mitochondria located in this region. A model is discussed in which the localization of mitochondria with respect to Ca2+ sources is the main determinant of their in situ Ca2+ uptake kinetics. Thus, in any given cell type mitochondria may be localized to suit the energy and metabolic demands of their physiological actions.
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              • Record: found
              • Abstract: not found
              • Article: not found

              Cellular mechanisms regulating [Ca2+]i smooth muscle.

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                Author and article information

                Journal
                The Journal of Physiology
                Wiley
                00223751
                January 1998
                January 1998
                September 22 2004
                : 506
                : 1
                : 109-125
                Article
                10.1111/j.1469-7793.1998.109bx.x
                f5b1f62f-6143-40d0-955b-1ba81ba7dc95
                © 2004

                http://doi.wiley.com/10.1002/tdm_license_1.1

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