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Domains of high Ca2+ beneath the plasma membrane of living A7r5 cells
No full textTheoretical models and indirect experimental observations predict that Ca2+ concentrations at the inner surface of the plasma membrane may reach, upon stimulation, values much higher than those of the bulk cytosol. In the past few years, we have shown that the Ca2+-sensitive photoprotein aequorin can be intracellularly targeted and utilized for specifically monitoring the [Ca2+] of various organelles. In this work, we extend this approach to the study of the cytoplasmic rim beneath the plasma membrane. We have constructed a new aequorin chimera by fusing the photoprotein with SNAP-25, a neuronal protein which is recruited to the plasma membrane after the post-translational addition of a lipid anchor. The SNAP-25-aequorin chimera, expressed in the rat aortic smooth muscle cell line A7r5, appears correctly sorted as revealed by immunocytochemistry. Using this probe, we demonstrate that the mean [Ca2+] of this cytoplasmic region ([Ca2+]pm) can reach values >10-fold higher than those of the bulk cytosol ([Ca2+]c) upon activation of Ca2+ influx through plasma membrane channels. In unstimulated cells, the mean [Ca2+]pm appears also to be higher than the bulk cytosol, presumably reflecting the existence of microdomains of high [Ca2+]
TARGETED CHIMERIC AEQUORINS - NEW TOOLS FOR STUDYING CA2+ HOMEOSTASIS AT THE SUBCELLULAR LEVEL
No full textPhotoprotein-mediated measurement of calcium ion concentration in mitochondria of living cells.
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NUCLEAR TARGETING OF AEQUORIN - A NEW APPROACH FOR MEASURING NUCLEAR CA2+ CONCENTRATION IN INTACT-CELLS
No full textWe here describe the measurement of nuclear Ca2+ concentration ([Ca2+]n) with targeted recombinant aequorin. Two aequorin chimeras have been constructed, composed of the Ca(2+)-sensitive photoprotein and two different portions of the glucocorticoid hormone receptor (GR). The shorter chimera (nuAEQ), which contains the nuclear localization signal (NLS) NL1 of GR, but lacks its hormone binding domain, HBD, is constitutively localized in the nucleus; the longer one (nu/cytAEQ), which contains both NLSs (NL1 + NL2) and the HBS of GR, is normally localized in the cytosol, but is translocated to the nucleus upon treatment with the hormone. When localized to the nucleus, both chimeras give the same estimates of [Ca2+]n, both at rest and upon stimulation with the InsP3 generating agonist histamine. The [Ca2+]n values appear very close, both at rest and upon stimulation, to those of the cytoplasm, measured with cytosolic recombinant aequorin, suggesting that, at least in this cell model, the nuclear membrane does not represent a major barrier to the diffusion of Ca2+ ions, and that the nucleus does not regulate its [Ca2+] independently from the cytosol
Domains of high Ca2+ beneath the plasma membrane of living A7r5 cells.
No full textTheoretical models and indirect experimental observations predict that Ca2+ concentrations at the inner surface of the plasma membrane may reach, upon stimulation, values much higher than those of the bulk cytosol. In the past few years, we have shown that the Ca2+-sensitive photoprotein aequorin can be intracellularly targeted and utilized for specifically monitoring the [Ca2+] of various organelles. In this work, we extend this approach to the study of the cytoplasmic rim beneath the plasma membrane. We have constructed a new aequorin chimera by fusing the photoprotein with SNAP-25, a neuronal protein which is recruited to the plasma membrane after the post-translational addition of a lipid anchor. The SNAP-25-aequorin chimera, expressed in the rat aortic smooth muscle cell line A7r5, appears correctly sorted as revealed by immunocytochemistry. Using this probe, we demonstrate that the mean [Ca2+] of this cytoplasmic region ([Ca2+]pm) can reach values >10-fold higher than those of the bulk cytosol ([Ca2+]c) upon activation of Ca2+ influx through plasma membrane channels. In unstimulated cells, the mean [Ca2+]pm appears also to be higher than the bulk cytosol, presumably reflecting the existence of microdomains of high [Ca2+]
Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). A critical evaluation.
No full textTargeted recombinant aequorins represent to date the most specific means of monitoring [Ca2+] in subcellular organelles (Rizzuto, R., Simpson, A. W. M., Brini, M., and Pozzan, T. (1992) Nature 358, 325-328; Brini, M., Murgia, M., Pasti, L., Picard, D., Pozzan, T., and Rizzuto, R. (1993) EMBO J. 12, 4813-4819; Kendall, J. M., Dormer, R. L., and Campbell, A. K. (1992) Biochem. Biophys. Res. Commun. 189, 1008-1016). Up until now, however, only limited attention has been paid to the use of recombinant photoproteins for measuring, in mammalian cells, the [Ca2+] in the cytoplasm, a compartment for which effective Ca2+ probes are already available. Here we describe this approach in detail, highlighting the advantages, under various experimental conditions, of using recombinant cytosolic aequorin (cytAEQ) instead of classical fluorescent indicators. We demonstrate that cytAEQ is expressed recombinantly at high levels in transiently transfected cell lines and primary cultures as well as in stably transfected clones, and we describe a simple algorithm for converting aequorin luminescence data into [Ca2+] values. We show that although fluorescent indicators at the usual intracellular concentrations (50-100 microM) are associated with a significant buffering of the [Ca2+]c transients, this problem is negligible with recombinantly expressed aequorin. The large dynamic range of the photoprotein also allows an accurate estimate of the large [Ca2+]c increases that are observed in some cell types such as neurons. Finally, cytAEQ appears to be an invaluable tool for measuring [Ca2+]c in cotransfection experiments. In particular, we show that when cotransfected with an alpha 1-adrenergic receptor (coupled to inositol 1,4,5-trisphosphate generation), cytAEQ faithfully monitors the subpopulation of cells expressing the receptor, whereas the signal of fura-2, at the population level, is dominated largely by that of the untransfected cells
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Rilmenidine activates postjunctianal alpha1- and alpha2-adrenoceptors in the canine saphenous vein
No full textExperiments were performed to determine the subtypes of alpha-adrenoceptors involved in the contraction induced by rilmenidine in isolated canine cutaneous veins. Rings of saphenous vein (without endothelium) were suspended for the recording of isometric force in physiological salt solution. All experiments were performed in the presence of propranolol (to antagonize beta-adrenoceptors), cocaine (to inhibit neuronal uptake) and hydrocortisone (to inhibit extraneuronal uptake). In the presence of rauwolscina (an alpha2-adrenegic blocker), rilmenidine caused concentration-dependent contractions which were inhibited by prazosin (nonselective alpha1-antagonist) and by (+)niguldipine (selective alpha(1A)-adrenergic antagonist), but not by (-)nigurdipine. After treatment with phenoxybenzamine (to alkylate alpha1-adrenoceptors), rilmenidine evoked contractions of the canine saphenous vein which were antagonized competitively by rauwolscine. The combination of rauwolscine and prazosin did not abolish contractions evoked by the highest concentrations of rilmenidine. Although binding experiments using 3H-idazoxan suggested the existence of a nonadrenergic binding site (around 20% of the total binding), contractile studies failed to demonstrate their involvement in the increases in tension evoked by rilmenidine. These experiments suggest that the contractions evoked by rilmenidine in isolated canine veins are mediated by both alpha(1A)- and alpha2-adrenoceptors
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