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Quenching of a proton gradient and concomitant increase of intragranular calcium in interstitial cells of Mytilus retractor muscle

Fluorescence microscopy and X-ray microanalysis

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Summary

The content of specific glio-interstitial granules in situ was studied in Mytilus retractor muscle using fluorescent probes and X-ray microanalysis. The granules readily take up the fluorescent monoamine dye acridine orange added to sea water (2.7×10-6 M) and appear red in fluorescence microscopy. The addition of ammonium chloride (10 mM) or various proton ionophores results in extinction of the granule fluorescence. In addition, a step-wise decrease in granule fluorescence is observed when the tissue is perfused with artificial sea water of decreasing pH. These granules thus appear to be acidic inside. The animals were maintained in artificial sea water containing 8.36 mM Ca2+ and 528.90 mM Na+, the ratio R=[Ca2+]0/[Na+]2 0 being thus equal to 3x10-5. Perfusions of the tissue with artificial sea water containing a higher calcium concentration (12.2 mM) and/or a higher [Ca2+]0/[Na+]2 0 ratio (R=4.5×10-5) result in a drastic reduction of the proton gradient, evidenced by a quenching of the acridine orange fluorescence. Under the same conditions, a significant increase of the total intragranular calcium concentration was demonstrated by quantitative X-ray micro-analysis of the tissue processed by quick freezing and freeze-substitution in the presence of oxalic acid. The fluorescence of the probe Fluo-3/AM, indicative of ionized calcium, is higher in the granules than in the surrounding cytoplasm; this suggests that calcium is accumulated in the granule against its concentration gradient. The acidic gradient of specific glio-interstitial cell granules could provide the energy needed for this calcium accumulation through a Ca2+/H+ exchange. These results are discussed with regard to the hypothesis that the glio-interstitial tissue can regulate pericellular calcium and/or hydrogen ion ioncentration in the vicinity of nerve and muscle cells.

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Authors and Affiliations

  1. Cytologie Expérimentale (URA 651 CNRS) and Centre Commun de Microscopie Appliquée, Université de Nice-Sophia Antipolis, Nice, France

    K. Maggio, E. Keicher, M. L. Hernandez-Nicaise & G. Nicaise

  2. Biologie Cellulaire Marine (URA 671 CNRS), Station Zoologique, Villefranche sur Mer, France

    I. Gillot

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  1. K. Maggio
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  2. E. Keicher
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  3. M. L. Hernandez-Nicaise
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  4. I. Gillot
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  5. G. Nicaise
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Maggio, K., Keicher, E., Hernandez-Nicaise, M.L. et al. Quenching of a proton gradient and concomitant increase of intragranular calcium in interstitial cells of Mytilus retractor muscle. Cell Tissue Res 262, 149–156 (1990). https://doi.org/10.1007/BF00327756

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