Aquaporins (AQPs), membrane water channel proteins expressed in various tissues and organs, serve in the transfer of water and small solutes across the membrane. We raised antibodies to AQPs using isoform-specific synthetic peptides and surveyed their expression in the rat nasal olfactory and respiratory mucosae. AQP1, AQP3, AQP4, and AQP5 were detected by immunohistochemical and immunoblotting analyses. AQP1 was expressed in the endothelial cells of blood vessels and the surrounding connective tissue cells in the olfactory and respiratory mucosae. AQP1 may be involved in water transfer across the blood vessel wall. In the olfactory epithelium, no AQP was detected in the olfactory sensory cells. Instead, AQP3 was abundant in the olfactory epithelium, where it was localized in the supporting cells and basal cells. Expression of AQP3 was mostly restricted to the basal cells in the respiratory epithelium. In marked contrast, AQP4 was abundant in the respiratory epithelium, but its abundance was limited to the basal cells in the olfactory epithelium. In the Bowman’s gland, AQP5 was localized in the apical membrane in the secretory acinar cells, whereas AQP3 and AQP4 were found in the basolateral membrane. Similar localization was seen in its duct cells. These results showed a distinct localization pattern for AQPs in the olfactory epithelium. AQP3 and AQP4 in the supporting cells and basal cells may play an important role in generating and maintaining the specific microenvironment around the olfactory sensory cells. AQP3, AQP4, and AQP5 in the Bowman’s gland may serve in the secretion to generate the microenvironment at the apical surface of the olfactory dendrites for odorant reception.

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Aluminium (Al) impairs the glutamate-nitric oxide-cGMP pathway and reduces the number of nitroxidergic neurons in the rat somatosensory cortex. To understand better the effect of the time of exposure, we monitored the effect of aluminium administration on nitroxidergic neurons, identified by NADPH-diaphorase (NADPH-d) or by nitric oxide synthase (NOS) staining, after 0.5, 1, 2, 3, 6 and 12 months of aluminium administration. Since neuropeptide Y (NPY) is known to be colocalised with nitric oxide synthase in cortical neurons, the aim of this work was to study the effects of Al administration on the cortical expression of NADPH-d, nNOS, and NPY. NADPH-d or NOS positive neurons were found scattered in the cortex where they constituted about 1% of all neurons. Double staining using NADPH-d and NPY showed that almost all nitroxidergic neurons were co-localised with NPY neurons (NADPH-d/NPY double stained neurons) whereas some neurons were stained only with NPY (NPY single stained neurons) ; these were more numerous than NADPH-d/NPY double stained neurons. Al significantly reduced NADPH-d and nNOS positive neurons in the cerebral cortex time dependently, with the greatest effect appearing after 3 months. Also measured was the integrated optical density (IOD) of nNOS positive neurons showing a significant decrease of NOS immunostaining even in the remaining NOS positive neurons. The double staining experiment exhibited a decrease in NADPH-d/NPY double stained neurons with an apparent increase in NPY single stained neurons; these then decreased after 6-12 months. On the whole, the results confirm that Al impairs nitroxidergic pathways time dependently; moreover, the transient increase in NPY single stained neurons from 1 to 3 months suggests that there is an intraneuronal down-regulation of NOS, without affecting neuronal viability. In addition, the decrease in the NPY system found at 6 and 12 months may indicate that Al affected nitroxidergic and NPY systems at different times.

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The requirement of a bone morphogenetic protein for the maintenance and stimulation of an osteoblast phenotype was examined using mouse MC3T3-E1 cell cultures. Cells expressed BMP-4 mRNA, which correlated with the stimulation of the osteoblast phenotype. The addition of a BMP-4 specific antibody reduced bone nodules, suggesting that BMP-4 is required for the osteogenic activity of osteoblasts in an autocrine manner. Exogenously added BMP-7 gradually decreased the expression of BMP-4 with a concurrent stimulation of the osteoblast phenotype. Exogenous BMP-7 can therefore substitute for endogenously produced BMP-4 acting as a paracrine factor on osteoblasts.The addition of 17β estradiol decreased BMP-4 expression but initiated synthesis of BMP-6 mRNA, an endocrine signal for osteoblasts, which also substituted for the lack of endogenous BMP-4, as evidenced by normal bone nodule formation. The addition of dexamethasone and parathyroid hormone did not affect the BMP-4 expression but induced transcripts for BMP-2 and BMP-3, respectively, suggesting that their effects on bone can be in part achieved via the BMP signaling. These experiments support the requirement of a BMP for osteoblast differentiation and function, demonstrating for the first time that a BMP can functionally substitute for another BMP in an autocrine/paracrine manner or mediate a response to an endocrine action on osteoblasts.

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We previously showed histologically that, in the rat, the cerebrospinal fluid drains from the subarachnoid space along the olfactory nerves to the nasal lymphatics and empties into the superficial and deep cervical lymph nodes. The present study was performed to investigate whether these lymph nodes play a role in the immune response of the central nervous system. For this purpose, keyhole limpet hemocyanin conjugated with fluorescein isothiocyanate (KLH-FITC) was administered into the subarachnoid space of the rat brain, and the time-kinetics and location of FITC and anti-FITC antibody forming cells in the cervical lymph nodes were studied histologically and immunohistochemically. FITC fluorescence was detected in superficial and deep cervical lymph nodes as well as the subarachnoid space and the nasal mucosa 2 h after FITC-KLH injection into the subarachnoid space. The specific antibody-forming cells first appeared in both the superficial and deep cervical lymph nodes on the 4th day after antigen administration although the reaction was more intense in the deep than in the superficial cervical lymph nodes. These cells were located in the medullary cords of the cervical lymph nodes. The number of antibody forming cells increased thereafter, reached a peak around the day 6, and then declined on day 10. These findings indicate that antigens introduced in the cerebrospinal fluid are drained into the cervical lymph nodes through the nasal lymphatics and initiate the antigen-specific immune response there. Thus, the cervical lymph nodes probably act as a monitoring site for cerebrospinal fluid and play a major role in the central nervous system immune response.

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Protease-activated receptors (PARs) mediate cellular responses to various proteases in numerous cell types, including smooth muscles and the endothelium of blood vessels. To clarify whether the stimulation of PARs induces responses in smooth muscle cells of cerebral arterioles, intracellular Ca²⁺([Ca²⁺]_i) dynamics and nitric oxide (NO) production during PARs stimulation were investigated in the rat cerebral arterioles by real-time confocal microscopy, since [Ca²⁺]_i and NO are both key factors in the maintenance of strain in blood vessels. Testicular arterioles were also investigated for comparison. In smooth muscle cells of small cerebral arterioles (< 50 μm in diameter), thrombin and PAR1-activating peptide (AP) induced an increase in [Ca²⁺]_i and contraction. The response to PAR1 activation was caused by Ca²⁺ mobilization from intracellular Ca²⁺ stores. Trypsin and PAR2-AP induced a decrease in [Ca²⁺]_i in the cells which was considered to be mediated by endothelium-derived NO and/or by promoting a Ca²⁺sequestration mechanism. PAR3- and 4-AP had little effect. In contrast to small cerebral arterioles, [Ca²⁺]_i dynamics in smooth muscle cells of large cerebral arterioles (< 150 μm in diameter) or testicular arterioles remained unchanged during PARs activation. The effects of PARs activation on the [Ca²⁺]_i dynamics and the contraction/relaxation of cerebral arterioles are also discussed in relation to the role of proteases in the regional tissue circulation of the brain.

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In the portal tract of the regenerating liver after partial hepatectomy, vascular and bile ductular remodeling takes place in response to the portal hyperdynamic state and parenchymal hyperplasia. In order to reveal phenotypical changes in the portal fibroblasts, we immunohistochemically investigated neural cell adhesion molecules (NCAM) and alpha smooth muscle actin (αSMA) expression and the ultrastructural changes in them during liver regeneration. In the control rat liver, portal fibroblasts were negative for both NCAM and αSMA. They became positive for both markers two days after partial hepatectomy, increased in staining intensity, reached a maximum at three to four days, then decreased, being still clearly positive at 14 days. Under an electron microscope, portal fibroblasts from the regenerating liver had larger amounts of cytoplasm and rough endoplasmic reticulum than those from the control liver; thus they might be activated. Additionally, periportal hepatic stellate cells in the regenerating liver were activated with αSMA, but without NCAM. The present study has demonstrated that portal fibroblasts express NCAM and αSMA in the regenerating liver after partial hepatectomy via transformation into myofibroblasts following reconstruction of the portal tracts.

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The present study introduces a method for obtaining three-dimensional images of native (i.e., unfixed) chromosomes by atomic force microscopy (AFM) in a liquid. Human metaphase chromosomes were isolated from a human lymphoblast-like cell line, K562, by the hexylene glycol procedure according to Wray and Stubble- field (1970), adsorbed on a silane-coated glass slide, and observed in a dynamic force mode (i.e., intermittent contact mode) of AFM in a hexylene buffer solution. In adequate operating conditions, the shape of chromosomes with paired chromatids was clearly and three-dimensionally observed by AFM. At high magnification, globular or fibrous structures about 50 nm thick could be found on the surface of each chromaid, implying that chromatin fibers were strongly wound or twisted in the chromatid. Thus, AFM imaging enabled the direct visualization of native chromosomes in a liquid at high resolution - which is com parable with that of scanning electron microscopy - and can serve to analyze the mechanism of chromosome condensation and separation in relation to the structure of chromosomes.

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