Iron in the brain is utilized for cellular respiration, neurotransmitter synthesis/degradation, and myelin formation. Iron, especially its ferrous form, also has the potential for catalyzing the Fenton reaction to generate highly cytotoxic hydroxyl radicals. The amount of iron in the brain must therefore be strictly controlled. In this study, we focused on the cellular and subcellular localizations of nonheme ferric (Fe(III)) and ferrous (Fe(II)) iron in the adult female rat brain using light and electron microscopic histochemistry. Although Fe(II) deposition was much less dominant than Fe(III), the brain contained iron in both forms. Among the cellular elements of the brain, oligodendrocytes were numerically the most prominent and heavily iron-storing cells. Pericapillary astrocytes and sporadic microglial cells also showed dense iron accumulation. Large neurons involved in the motor system were relatively strongly iron-positive. Subcellularly, Fe(III) and Fe(II) were mainly localized in lysosomes, and occasionally in the cytosol and mitochondria. Furthermore, capillary endothelial cells had Fe(III)-positive reactions in lysosomes and the cytosol, with Fe(II)-positive reactions on the luminal membrane. With advancing age, both Fe(III) and Fe(II) became more extensively distributed and accumulated more numerously in oligodendrocytes and astrocytes. These findings suggest that age-related increases in Fe(II) accumulation may raise the risk of tissue damage in the normal brain.

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The tracheal epithelium can be induced to move as a cellular sheet by heterotopic transplantation, which offers the opportunity to observe migrating cells as a group in an in vivo environment. We therefor investigated the ultrastructural characteristics of migrating tracheal epithelial cells with special reference to the moving front using this transplantation. The migrating epithelial cells underwent squamous metaplasia and lost their differentiated characteristics such as cilia or secretory granules. Several unique observations were made concerning the mechanism of mobility: one is that epithelial cells in the front were elongated in a direction perpendicular to the course of movement, different from previous reports in vitro. The second is that lamellipodia, which are regarded as the major locomotive machinery in the adult wound epithelium, did not make up the major part of the front; the major portion of the anterior fringe of the moving front was usually smooth and gently curved, and actin cables parallel to the elongated cells were observed by confocal laser microscopy, indicating that the purse-string mechanism of epithelial wound healing takes place. The third finding is that the cells in the front had irregular bleb-like structures on their antero-basal surface, which were formed even in the portion where the cells did not attach to the matrix. Few organelles were recognized in these structures. From their location, one might propose that these bleb-like structures play a role in the recognition of the substrate and thus the movement of the cell sheet.

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Dipyridamole, an inhibitor of adenosine uptake as well as a cGMP phosphodiesterase inhibitor, is commonly used in prophylactic therapy for patients with angina pectoris. However, the effects of dipyridamole on systemic blood vessels, especially on the peripheral vascular system, are not well understood. Therefore, the effect of dipyridamole on ATP-induced arteriole contraction was examined with special reference to intracellular Ca²⁺ concentration ([Ca²⁺]_i) using real-time confocal microscopy. In cases of 0.1-10μM range, dipyridamole induced only slight [Ca²⁺]_i decreases in smooth muscle cells of both testicular and cerebral arterioles. However, 100μM dipyridamole induced substantial [Ca²⁺]_i decreases in the cells. In the presence of 10μM dipyridamole, changes in ATP-induced [Ca²⁺]_i were found to be inhibited in smooth muscle cells of testicular arterioles but not in those of cerebral arterioles. In addition, α, β-methylene ATP-induced [Ca²⁺]_i increases in testicular arteriole smooth muscle cells were also partially inhibited in the presence of dipyridamole. When testicular arterioles were perfused with dipyridamole, no increases in nitric oxide levels were detected. High levels of K⁺ induced a [Ca²⁺]_i increase in testicular arterioles that was also partially inhibited by dipyridamole. In the presence of substances that affect protein kinase A or G, ATP-induced [Ca²⁺]_i was not completely inhibited. These findings suggest that dipyridamole may act not only as an inhibitor of adenosine uptake and as a cGMP phosphodiesterase inhibitor, but also as a calcium channel blocker in arteriole smooth muscle cells.

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Regulation of the intracellular calcium ion concentration ([Ca²⁺]_i) is critical, because calcium signaling controls diverse and vital cellular processes such as secretion, proliferation, division, gene transcription, and apoptosis. Store-operated calcium entry (SOCE) is the main mechanism through which non-excitable cells replenish and thus maintain this delicate balance. There is limited evidence which indicates that SOCE may be inhibited during mitosis, and the mechanisms leading to the presumed inhibition has not been elucidated. In the present study, we examined and compared the [Ca²⁺]_i dynamics of COS-7 cells in mitotic and non-mitotic phases with special reference paid to SOCE. Laser scanning confocal microscopy to monitor [Ca²⁺]_i dynamics revealed that SOCE was progressively inhibited in mitosis and became virtually absent during the metaphase. We used various cytoskeletal modifying drugs and immunofluorescence to assess the contribution of microtubule and actin filaments in SOCE signaling. Nocodazole treatment caused microtubule reorganization and retraction from the cell periphery that mimicked the natural mitotic microtubule remodeling that was also accompanied by SOCE inhibition. Short exposure to paclitaxel, a microtubule-stabilizing drug, bolstered SOCE, whereas long exposure resulted in microtubule disruption and SOCE inhibition. Actin-modifying drugs did not affect SOCE. These findings indicate that mitotic microtubule remodeling plays a significant role in the inhibition of SOCE during mitosis.

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The expression of stress-responsive proteins, such as nestin and a 27-kDa heat-shock protein (HSP27), was immunohistochemically examined in order to demonstrate glial responses in the rat olfactory bulb following sensory deprivation. At 3 days to 1 week after sensory deprivation, numerous nestin-expressing cells appeared within the glomerulus of the olfactory bulb. These cells were regarded as reactive astrocytes since they were immunoreactive for glial fibrillary acidic protein and showed hypertrophic features. The glomeruli, in which nestin-immunoreactive astrocytes were localized, were filled with degenerating terminals of olfactory receptor neurons and migrated microglia. A small population of nestin-immunoreactive cells was positive for a proliferating cell marker, Ki67 (8.0-9.7% at 3 days; 3.1 - 5.0% at 1 week). At 3 weeks, nestin-immunoreactive astrocytes were occasionally detected. At 6 weeks, when the olfactory receptor neurons had completely recovered, no nestin-immunoreactive astrocytes were detected. HSP 27 was also expressed within the glomerular astrocytes and showed a similar spatiotemporal expression pattern to nestin. The present study suggests that reactive astrocytes may be involved in axonal regeneration and synaptic remodeling in the olfactory system, through the recapitulation of developmentally regulated proteins, such as nestin and HSP27.

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