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. 2003 Mar 1;23(5):1580-3.
doi: 10.1523/JNEUROSCI.23-05-01580.2003.

Calcium-dependent exocytosis of atrial natriuretic peptide from astrocytes

Mojca Krzan  1 , Matjaz Stenovec, Marko Kreft, Tina Pangrsic, Sonja Grilc, Philip G Haydon, Robert Zorec
Affiliations

Calcium-dependent exocytosis of atrial natriuretic peptide from astrocytes

Mojca Krzan et al. J Neurosci. .

Abstract

Astrocytes are non-neuronal cells in the CNS, which, like neurons, are capable of releasing neuroactive molecules. However, the mechanism of release is ill defined. In this study, we investigated the mechanism of release of atrial natriuretic peptide (ANP) from cultured cortical astrocytes by confocal microscopy. To study the discharge of this hormone, we transfected astrocytes with a construct to express pro-ANP fused with the emerald green fluorescent protein (ANP.emd). The transfection of cells with ANP.emd resulted in fluorescent puncta in the cytoplasm that represent secretory organelles. If ANP is released by exocytosis, in which the vesicle fuses with the plasma membrane, then the total intensity of the green fluorescing probe should decrease, whereas the vesicle membrane is incorporated into the plasma membrane. To monitor exocytosis, we labeled the membrane with the fluorescent styryldye FM 4-64, a reporter of cumulative exocytosis. The application of ionomycin to elevate cytoplasmic [Ca(2+)] increased the fluorescence intensity of FM 4-64, whereas that of ANP.emd decreased. These effects were not observed in the absence of extracellular Ca(2+), suggesting that ANP is released by regulated Ca(2+)-dependent exocytosis from astrocytes.

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Figures

Fig. 1.
Fig. 1.
Confocal image of cultured cortical astrocyte transfected with an ANP.emd construct. Green fluorescent puncta are individual secretory granules that store ANP.emd (top). The same astrocyte is also observed under differential interference contrast optics (bottom). Scale bar, 2 μm.
Fig. 2.
Fig. 2.
Ionomycin causes exocytotic release of ANP.emd. Confocal images of double-labeled astrocyte before (left; 0 sec) and 60 sec after (right) bath application of ionomycin (10 μm) in the presence of 2 mm Ca2+ in the bath solution.Top, ANP.emd fluorescence. Bottom, FM 4-64 stain. Note the decreased number of green fluorescent puncta and a large increase in the surface membrane area indicated by the intense FM 4-64 fluorescence staining after 60 sec. Scale bars, 5 μm.
Fig. 3.
Fig. 3.
Ca2+-dependent release of ANP from cultured astrocytes. Normalized FM 4-64 fluorescence increase (left) and ANP.emd fluorescence decrease (right) obtained in a single astrocyte after the application of 10 μm ionomycin in Ca2+-free (top) and Ca2+-containing (2 mm) extracellular solution (bottom). The inset shows the overlay of fluorescence changes obtained with ANP.emd (filled symbols) and FM 4-64 (open symbols), showing no delay in the onset of ionomycin-induced fluorescence changes.
Fig. 4.
Fig. 4.
Mean relative change of FM 4-64 (left) and ANP.emd (right) fluorescence obtained in different cells stimulated with 10 μmionomycin in extracellular solution containing 2 mmCa2+ (n = 7) and Ca2+-free extracellular solution (n = 5). Asterisks indicate a statistically significant difference in the change in FM 4-64 and ANP.emd fluorescence change in the presence or absence of extracellular Ca2+ (FM 4-64, p = 0.0018; ANP.emd, p = 0.036; t test).
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