Claudin-5 is a protein component of many endothelial tight junctions, including those at the blood-brain barrier, a barrier that limits molecular exchanges between the central nervous system and the circulatory system. To test the contribution of claudin-5 to this barrier function of tight junctions, we expressed murine claudin-5 in Madin-Darby canine kidney II cells. The result was a fivefold increase in transepithelial resistance in claudin-5 transductants and a reduction in conductance of monovalent cations. However, the paracellular flux of neither neutral nor charged monosaccharides was significantly changed in claudin-5 transductants compared to controls. Therefore, expression of claudin-5 selectively decreased the permeability to ions. Additionally, site-directed mutations of particular amino acid residues in the first extracellular domain of claudin-5 altered the properties of the tight junctions formed in response to claudin-5 expression. In particular, the conserved cysteines were crucial: mutation of either cysteine abolishted the ability of claudin-5 to increase transepithelial resistance, and mutation of Cys 64 strikingly increased the paracellular flux of monosaccharides. These new insights into the functions of claudin-5 at the molecular level in tight junctions may account for some aspects of the blood-brain barrier's selective permeability.
Brain water transport is poorly understood at the molecular level, and marked changes occur during brain development. As the aquaporin-4 (AQP4) water channel protein is abundant in brain, the expression levels and subcellular distribution of this protein were examined during postnatal development. This study focused on the cerebellum, which showed the same pattern of AQP4 development as the rest of the brain. Semiquantitative immunoblotting revealed very low levels of AQP4 in the first postnatal week. A pronounced increase was noted in the second week, from 2% of adult level at postnatal day 7 (PN7) to 25% at PN14. At PN1 and PN3 immunofluorescence microscopy revealed weak labelling, mainly in radial processes (Bergmann fibres) and at the pial surface. Between PN7 and PN14 the labelling underneath the pia showed a strong increase, and immunoreactivity also appeared around blood vessels throughout the cerebellum. High-resolution immunogold electron microscopy revealed that the subpial and perivascular labelling was restricted to glial end feet, notably to those plasma membrane domains that were apposed to the basal laminae. At no stage was there any evidence of neuronal AQP4 labelling, and AQP1, -2, -3 and -5 proteins were not detected in the neuropil. Riboprobes to AQP4 mRNA produced a particularly strong in situ hybridization signal in glial cells between PN7 and PN14, corresponding to the stage of the most rapid increase of AQP4 protein. The time course and pattern of AQP4 expression suggests that this aquaporin plays an important role in brain water and K+ homeostasis from the second week of development.
Abstract. Clinical studies have shown that aquaporin-2 (AQP2), the vasopressin-regulated water channel, is excreted in the urine, and that the excretion increases in response to vasopressin. However, the cellular mechanisms involved in AQP2 excretion are unknown, and it is unknown whether the excretion correlates with AQP2 levels in kidney or levels in the apical plasma membrane. The present study was undertaken to clarify these issues. Immunoblotting of rat urine samples revealed significant excretion of AQP2, whereas AQP3, being a basolateral aquaporin in the same cells, was undetectable. Thus, there was a nonproportional excretion of AQP2 and AQP3 (compared with kidney levels), indicating that AQP2 is excreted predominantly via a selective apical pathway and not by whole cell shedding. Urinary AQP2 was associated with small vesicles, membrane fragments, and multivesicular bodies as determined by immunoelectron microscopy and negative staining techniques. In rats with normal water supply, daily urinary excretion of AQP2 was 3.9 ± 0.9% (n = 6) of total kidney expression. Treatment with desmopressin acetate subcutaneously caused a fourfold increase in urinary excretion of AQP2 during 8 h. Forty-eight hours of thirsting, known to increase endogenous vasopressin secretion, resulted in a three-fold increase in kidney AQP2 levels but urinary excretion increased ninefold to 15 ± 3% (n = 6) of AQP2 in kidney of thirsted rats. Moreover, rats that were thirsted for 48 h and subsequently allowed free access to water for 24 h produced a decrease in urinary AQP2 excretion to 38 ± 15% (n = 6) of that during thirsting. In Brattleboro rats or lithium-treated normal rats completely lacking vasopressin action, and hence having extremely low levels of AQP2 in the apical plasma membrane, AQP2 was undetectable in urine. Thus, conditions with known altered vasopressin levels and altered levels of AQP2 in the apical plasma membrane were associated with corresponding major changes in AQP2 urine excretion. In contrast, in such conditions, kidney AQP2 levels and urinary AQP2 excretion did not show a proportional relationship.
The postnatal development of total, type-A and type-B monoamine oxidase (MAO) in the brain stem, forebrain and cerebellum, determined with preferred substrates or selective inhibitors, were found to follow different patterns. In the brain regions, MAO-A activity reached adult levels in the brain stem first, followed by the forebrain and cerebellum, while MAO-B reached adult levels in these regions at about the same time and later in postnatal life. On the other hand, both MAO-A and B activities were almost fully developed in the newborn liver. Moreover, total and type-A, but not type-B, showed a caudal-to-rostral sequence of biochemical maturation in the brain. The spatiotemporal pattern of differentiation of type-A and type-B activities in the brain tends to support the classification of brain MAO into two distinct isoenzymic forms.
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