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      Calcium Imaging of Murine Thoracic Aorta Endothelium by Confocal Microscopy Reveals Inhomogeneous Distribution of Endothelial Cells Responding to Vasodilator Agents

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          The aim of the study was to assess, in intact murine thoracic aorta in vitro, the distribution of endothelial cells responsive to endothelium-dependent vasodilators ACh, ATP, bradykinin and substance P, using laser line confocal microscopy in combination with two Ca<sup>2+</sup> fluorescent dyes, Fluo-4 and Fura-red. We observed that 82 ± 3% of endothelial cells responded to ATP, 33 ± 5% to Ach, whereas less than 3% of them responded to bradykinin or substance P. In order to determine whether the findings of pharmacological tests agree with confocal microscopy data, endothelium-dependent vasodilators induced relaxation was evaluated using isometric tension measurement. We show a marked correlation between a higher number of activated endothelial cells, using confocal microscopy, and a greater degree of endothelium-dependent relaxation using isometric tension measurement (p = 0.00286). Our results suggest that endothelial cells responding to endothelium-dependent vasodilators are not homogeneously distributed in intact murine thoracic aorta. This could be due to nonhomogeneous distribution of surface receptors or to differences in post-receptor coupling mechanisms.

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          Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes.

          We have developed fluo-4, a new fluorescent dye for quantifying cellular Ca2+ concentrations in the 100 nM to 1 microM range. Fluo-4 is similar in structure and spectral properties to the widely used fluorescent Ca(2+)-indicator dye, fluo-3, but it has certain advantages over fluo-3. Due to its greater absorption near 488 nm, fluo-4 offers substantially brighter fluorescence emission when used with excitation by argon-ion laser or other sources in conjunction with the standard fluorescein filter set. In vitro, fluo-4 exhibited high fluorescence emission, a high rate of cell permeation, and a large dynamic range for reporting [Ca2+] around a Kd(Ca2+) of 345 nM. We have also developed several Ca(2+)-indicators related to fluo-4 having lower affinities for Ca2+ that are useful in cellular studies requiring quantification of higher [Ca2+]. In a variety of physiological studies of live cells, fluo-4 labeled cells more brightly than did fluo-3, when challenged with procedures designed to elevate calcium levels. Fluo-4 is well suited for photometric and imaging applications that make use of confocal laser scanning microscopy, flow cytometry, or spectrofluorometry, or in fluorometric high-throughput microplate screening assays. Because of its higher fluorescence emission intensity, fluo-4 can be used at lower intracellular concentrations, making its use a less invasive practice.
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            A comparison of fluorescent Ca2+ indicator properties and their use in measuring elementary and global Ca2+ signals.

            Quantifying the magnitude of Ca2+ signals from changes in the emission of fluorescent indicators relies on assumptions about the indicator behaviour in situ. Factors such as osmolarity, pH, ionic strength and protein environment can affect indicator properties making it advantageous to calibrate indicators within the required cellular or subcellular environment. Selecting Ca2+ indicators appropriate for a particular application depends upon several considerations including Ca2+ binding affinity, dynamic range and ease of loading. These factors are usually best determined empirically. This study describes the in-situ calibration of a number of frequently used fluorescent Ca2+ indicators (Fluo-3, Fluo-4, Calcium Green-1, Calcium Orange, Oregon Green 488 BAPTA-1 and Fura-Red) and their use in reporting low- and high-amplitude Ca2+ signals in HeLa cells. All Ca2+ indicators exhibited lower in-situ Ca2+ binding affinities than suggested by previously published in-vitro determinations. Furthermore, for some of the indicators, there were significant differences in the apparent Ca2+ binding affinities between nuclear and cytoplasmic compartments. Variation between indicators was also found in their dynamic ranges, compartmentalization, leakage and photostability. Overall, Fluo-3 proved to be the generally most applicable Ca2+ indicator, since it displayed a large dynamic range, low compartmentalization and an appropriate apparent Ca2+ binding affinity. However, it was more susceptible to photobleaching than many of the other Ca2+ indicators.
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              Elevation of intracellular calcium in smooth muscle causes endothelial cell generation of NO in arterioles.

              It is well known that vascular smooth muscle tone can be modulated by signals arising in the endothelium (e.g., endothelium-derived relaxing factor, endothelium-derived hyperpolarizing factor, and prostaglandins). Here we show that during vasoconstriction a signal can originate in smooth muscle cells and act on the endothelium to cause synthesis of endothelium-derived relaxing factor. We studied responses to two vasoconstrictors (phenylephrine and KCl) that act by initiating a rise in smooth muscle cell intracellular Ca2+ concentration ([Ca2+]i) and exert little or no direct effect on the endothelium. Fluo-3 was used as a Ca2+ indicator in either smooth muscle or endothelial cells of arterioles from the hamster cheek pouch. Phenylephrine and KCl caused the expected rise in smooth muscle cell [Ca2+]i that was accompanied by an elevation in endothelial cell [Ca2+]i. The rise in endothelial cell [Ca2+]i was followed by increased synthesis of NO, as evidenced by an enhancement of the vasoconstriction induced by both agents after blockade of NO synthesis. The molecule involved in signal transmission from smooth muscle to endothelium is as yet unknown. However, given that myoendothelial cell junctions are frequent in these vessels, we hypothesize that the rise in smooth muscle cell Ca2+ generates a diffusion gradient that drives Ca2+ through myoendothelial cell junctions and into the endothelial cells, thereby initiating the synthesis of NO.

                Author and article information

                J Vasc Res
                Journal of Vascular Research
                S. Karger AG
                June 2002
                22 August 2008
                : 39
                : 3
                : 260-267
                Departments of aInternal Medicine, Centre Hospitalier Universitaire de Rouen-Boisguillaume, Rouen, France, and bZoology and Animal Biology, University of Geneva, Sciences III, Geneva, Switzerland
                63691 J Vasc Res 2002;39:260–267
                © 2002 S. Karger AG, Basel

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                Page count
                Figures: 5, References: 36, Pages: 8
                Research Paper


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