Ca2+ signaling in endothelial cells stimulated by bradykinin: Ca2+ measurement in the mitochondria and the cytosol by confocal microscopy.
In this study we have monitored the change of intracellular Ca2+ concentrations in the cytosol ([Ca2+]c) and the mitochondria ([Ca2+]m) of single bovine endothelial cells following treatment with bradykinin (BK). Using laser scanning confocal microscopy, we have found that the Ca2+ indicator, Fluo-3, is compartmentalized in the mitochondria of endothelial cells loaded with Fluo-3/AM. After BK stimulation, the pattern of Ca2+ increase in the cytosol is different from that in the mitochondria. The amplitude of the Ca2+ rise in the mitochondria is higher than that in the cytosol. Further analysis using rapid scanning measurements indicates that the [Ca2+]c increase is very fast after BK addition and reaches a maxima level within 400 ms. In contrast, the [Ca2+]m increase appears to be biphasic with an initial rapid increase (concomitant with the [Ca2+]c increase) followed by a slower [Ca2+]m increase before reaching a maximal level (within 5 s of BK treatment). The differential Ca2+ signaling pattern between the cytosol and the mitochondria suggests that the intracellular Ca2+ concentrations needed to regulate various Ca(2+)-dependent enzymes located in these two compartments are different during BK-induced endothelial cell activation.
Donnadieu
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Department of Cell Biology and Anatomy, University of Miami Medical School, Florida 33101, USA.
Bourguignon
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D000814:Aniline Compounds; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology*; D002417:Cattle; D002451:Cell Compartmentation; D003600:Cytosol / Q000737:chemistry; D004730:Endothelium, Vascular / Q000166:cytology* / Q000648:ultrastructure; D005456:Fluorescent Dyes; D007700:Kinetics; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000737:chemistry; D015398:Signal Transduction / Q000187:drug effects; D014966:Xanthenes
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D014966:Xanthenes; C059715:Fluo-3; D001920:Bradykinin; D002118:Calcium