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      Cardiac Microvascular Endothelial Cells Express a Functional Ca 2+-Sensing Receptor

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          The mechanism whereby extracellular Ca<sup>2+</sup> exerts the endothelium-dependent control of vascular tone is still unclear. In this study, we assessed whether cardiac microvascular endothelial cells (CMEC) express a functional extracellular Ca<sup>2+</sup>-sensing receptor (CaSR) using a variety of techniques. CaSR mRNA was detected using RT-PCR, and CaSR protein was identified by immunocytochemical analysis. In order to assess the functionality of the receptor, CMEC were loaded with the Ca<sup>2+</sup>-sensitive fluorochrome, Fura-2/AM. A number of CaSR agonists, such as spermine, Gd<sup>3+</sup>, La<sup>3+</sup> and neomycin, elicited a heterogeneous intracellular Ca<sup>2+</sup> signal, which was abolished by disruption of inositol 1,4,5-trisphosphate (InsP<sub>3</sub>) signaling and by depletion of intracellular stores with cyclopiazonic acid. The inhibition of the Na<sup>+</sup>/Ca<sup>2+</sup> exchanger upon substitution of extracellular Na<sup>+</sup> unmasked the Ca<sup>2+</sup> signal triggered by an increase in extracellular Ca<sup>2+</sup> levels. Finally, aromatic amino acids, which function as allosteric activators of CaSR, potentiated the Ca<sup>2+</sup> response to the CaSR agonist La<sup>3+</sup>. These data provide evidence that CMEC express CaSR, which is able to respond to physiological agonists by mobilizing Ca<sup>2+</sup> from intracellular InsP<sub>3</sub>-sensitive stores.

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          Most cited references 24

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          Extracellular calcium sensing and signalling.

           M Hofer,  Micah Brown (2003)
          Ca2+ is well established as an intracellular second messenger. However, the molecular identification of a detector for extracellular Ca2+--the extracellular calcium-sensing receptor--has opened up the possibility that Ca2+ might also function as a messenger outside cells. Information about the local extracellular Ca2+ concentration is conveyed to the interior of many cell types through this unique G-protein-coupled receptor. Here, we describe new emerging concepts concerning the signalling function of extracellular Ca2+, with particular emphasis on the extracellular calcium-sensing receptor.
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            Submaximal stimulation of porcine endothelial cells causes focal Ca2+ elevation beneath the cell membrane.

            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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              Cloning and functional expression of a rat kidney extracellular calcium/polyvalent cation-sensing receptor.

              The maintenance of a stable extracellular concentration of ionized calcium depends on the integrated function of a number of specialized cells (e.g., parathyroid and certain kidney epithelial cells). We recently identified another G protein-coupled receptor (BoPCaRI) from bovine parathyroid that responds to changes in extracellular Ca2+ within the millimolar range and provides a key mechanism for regulating the secretion of parathyroid hormone. Using an homology-based strategy, we now report the isolation of a cDNA encoding an extracellular Ca2+/polyvalent cation-sensing receptor (RaKCaR) from rat kidney. The predicted RaKCaR protein shares 92% identity with BoPCaR1 receptor and features a seven membrane-spanning domain, characteristic of the G protein-coupled receptors, which is preceded by a large hydrophilic extracellular NH2 terminus believed to be involved in cation binding. RaKCaR cRNA-injected Xenopus oocytes responded to extracellular Ca2+, Mg2+, Gd3+, and neomycin with characteristic activation of inositol phospholipid-dependent, intracellular Ca(2+)-induced Cl- currents. In rat kidney, Northern analysis revealed RaKCaR transcripts of 4 and 7 kb, and in situ hybridization showed localization primarily in outer medulla and cortical medullary rays. Our results provide important insights into the molecular structure of an extracellular Ca2+/polyvalent cation-sensing receptor in rat kidney and provide another basis on which to understand the role of extracellular divalent cations in regulating kidney function in mineral metabolism.

                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                December 2008
                24 June 2008
                : 46
                : 1
                : 73-82
                aDepartment of Biomedicine, School of Medicine, Benemèrita Universidad Autònoma de Puebla, Puebla, Mexico; bDepartment of Cellular and Molecular Physiological and Pharmacological Sciences, University of Pavia, and cDepartment of Experimental Medicine, Section of Human Physiology, Pavia, dDepartment of Structural and Functional Biology, Federico II University, Naples, and eUnit of Histology, Department of Biomedical Sciences and Biotechnology, University of Brescia, Brescia, Italy; fFacultad de Ciencias Exactas, Università de La Plata, La Plata, Argentina
                140677 J Vasc Res 2009;46:73–82
                © 2008 S. Karger AG, Basel

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                Page count
                Figures: 7, References: 40, Pages: 10
                Research Paper


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