mitotracker-green-fm and diacetyldichlorofluorescein

Laser irradiation-induced phototoxicity has been intensively applied in clinical photodynamic therapy for the treatment of a variety of tumors. However, the precise laser damage sites as well as the underlying mechanisms at the subcellular level are unknown. Using a mitochondrial fluorescent marker, MitoTracker Green, severe mitochondrial swelling was noted in laser-irradiated rat brain astrocytes. Nucleus condensation and fragmentation revealed by propidium iodide nucleic acid staining indicated that laser-irradiated cells died from apoptosis. Using an intracellular reactive oxygen species (ROS) fluorescent dye, 2',7'-dichlorofluorescin diacetate, heterogeneous distribution of ROS inside astrocytes was observed after laser irradiation. The level of ROS in the mitochondrial compartment was found to be higher than in other parts of the cell. With another ROS fluorescent dye, dihydrorhodamine-123, and time-lapse laser scanning confocal microscopy, a substantial increase in mitochondrial ROS (mROS) was visualized in visible laser-irradiated astrocytes. The antioxidants melatonin and vitamin E largely attenuated laser irradiation-induced mROS formation and prevented apoptosis. Cyclosporin A (CsA), a mitochondrial permeability transition (MPT) blocker, did not prevent visible laser irradiation-induced mROS formation and apoptosis. In conclusion, mROS formation contributes significantly to visible laser irradiation-induced apoptosis via an MPT-independent pathway.

Topics: Aldehydes; Animals; Antioxidants; Apoptosis; Astrocytes; Brain; Cells, Cultured; Fluoresceins; Lasers; Melatonin; Microscopy, Confocal; Mitochondria; Propidium; Rats; Reactive Oxygen Species; Vitamin E

Cytokine-induced lung expression of the endothelial cell (EC) leukocyte receptor P-selectin initiates leukocyte rolling. To understand the early EC signaling that induces the expression, we conducted real-time digital imaging studies in lung venular capillaries. To compare receptor- vs nonreceptor-mediated effects, we infused capillaries with respectively, TNF-alpha and arachidonate. At concentrations adjusted to give equipotent increases in the cytosolic Ca(2+), both agents increased reactive oxygen species (ROS) production and EC P-selectin expression. Blocking the cytosolic Ca(2+) increases abolished ROS production; blocking ROS production abrogated P-selectin expression. TNF-alpha, but not arachidonate, released Ca(2+) from endoplasmic stores and increased mitochondrial Ca(2+). Furthermore, Ca(2+) depletion abrogated TNF-alpha responses partially, but arachidonate responses completely. These differences in Ca(2+) mobilization by TNF-alpha and arachidonate were reflected in spatial patterning in the capillary in that the TNF-alpha effects were localized at branch points, while the arachidonate effects were nonlocalized and extensive. Furthermore, mitochondrial blockers inhibited the TNF-alpha- but not the arachidonate-induced responses. These findings indicate that the different modes of Ca(2+) mobilization determined the spatial patterning of the proinflammatory response in lung capillaries. Responses to TNF-alpha revealed that EC mitochondria regulate the proinflammatory process by generating ROS that activate P-selectin expression.

Topics: Aldehydes; Animals; Calcium; Calcium Signaling; Capillaries; Endothelium, Vascular; Fluoresceins; Fluorescent Dyes; Heterocyclic Compounds, 3-Ring; Inflammation; Lung; Male; Microscopy, Confocal; Mitochondria; P-Selectin; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Tumor Necrosis Factor-alpha; Veins; Xanthenes