Aquaporin-5 water channel in lipid rafts of rat parotid glands.
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Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. The activation of M3 muscarinic acetylcholine receptors (mAChRs) or alpha1-adrenoceptors on the salivary glands induces salivary fluid secretion. AQP5 localizes in lipid rafts and activation of the M3 mAChRs or alpha1-adrenoceptors induced its translocation together with the lipid rafts to the APM in the interlobular ducts of rat parotid glands. This review focuses on the mechanisms of AQP5 translocation together with lipid rafts to the APM in the interlobular duct cells of parotid glands of normal rats and the impairment of AQP5 translocation in diabetes and senescence. (+info)
Heparin and fibroblast growth factors affect surfactant protein gene expression in type II cells.
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The stimulation and maintenance of the pulmonary alveolar type II cell's capacity to biosynthesize, store, and secrete surfactant proteins (SPs) are modulated to a great extent by growth factors, extracellular matrix (ECM) components, and hormones. It is possible that differences in ECM composition, as exist between type I and II cells normally or as might occur with excessive cell surface shedding during inflammation or injury states, may specifically alter SP expression. Here, isolated type II cells were exposed to the model sulfated ECM heparin; desulfated heparin; and/or fibroblast growth factor (FGF)-1, -2, or -7 for 24 h to examine by quantitative real-time polymerase chain reaction their effects on SP gene expression. Aquaporin 5 (AQP-5) gene expression was also examined as a phenotypic marker for the type I cell. SP-B mRNA abundance was increased 4- to 8-fold by all three FGFs. Heparin at low concentrations (5 microg/ml) or desulfated heparin at high concentrations (500 microg/ml) enhanced the effects of FGF-2 and -7, while high heparin concentrations (500 microg/ml) were inhibitory. In contrast, SP-B mRNA abundance was increased by heparin in a dose- and sulfation-dependent manner when used in combination with FGF-1. SP-C and AQP-5 mRNA levels were increased by heparin alone in a dose- and sulfation-dependent manner, while all FGFs lacked effect on SP-C or AQP-5 mRNA levels. These data indicate that heparin can be stimulatory to SP gene expression depending on concentration, degree of sulfation, and surrounding FGF environment, and that heparin plays a significant role in modulating alveolar epithelial cell phenotype in vitro. (+info)
Estrogen-induced proliferation of uterine epithelial cells is independent of estrogen receptor alpha binding to classical estrogen response elements.
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Acting via the estrogen receptor (ER), estradiol exerts pleomorphic effects on the uterus, producing cyclical waves of cellular proliferation and differentiation in preparation for embryo implantation. In the classical pathway, the ER binds directly to an estrogen response element to activate or repress gene expression. However, emerging evidence supports the existence of nonclassical pathways in which the activated ER alters gene expression through protein-protein tethering with transcription factors such as c-Fos/c-Jun B (AP-1) and Sp1. In this report, we examined the relative roles of classical and nonclassical ER signaling in vivo by comparing the estrogen-dependent uterine response in mice that express wild-type ERalpha, a mutant ERalpha (E207A/G208A) that selectively lacks ERE binding, or ERalpha null. In the compound heterozygote (AA/-) female, the nonclassical allele (AA) was insufficient to mediate an acute uterotrophic response to 17beta-estradiol (E2). The uterine epithelial proliferative response to E2 and 4-hydroxytamoxifen was retained in the AA/-females, and uterine luminal epithelial height increased commensurate with the extent of ERalpha signaling. This proliferative response was confirmed by 5-bromo-2'-deoxyuridine incorporation. Microarray experiments identified cyclin-dependent kinase inhibitor 1A as a nonclassical pathway-responsive gene, and transient expression experiments using the cyclin-dependent kinase inhibitor 1A promoter confirmed transcriptional responses to the ERalpha (E207A/G208A) mutant. These results indicate that nonclassical ERalpha signaling is sufficient to restore luminal epithelial proliferation but not other estrogen-responsive events, such as fluid accumulation and hyperemia. We conclude that nonclassical pathway signaling via ERalpha plays a critical physiologic role in the uterine response to estrogen. (+info)
Molecular and cellular characterization of a new aquaporin, AQP-x5, specifically expressed in the small granular glands of Xenopus skin.
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A new toad aquaporin (AQP) cDNA was cloned from a cDNA library constructed from the ventral skin of Xenopus laevis. This AQP (Xenopus AQP-x5) consisted of 273 amino acid residues with a high sequence homology to mammalian AQP5. The predicted amino acid sequence contained the two conserved Asn-Pro-Ala motifs found in all major intrinsic protein (MIP) family members and six putative transmembrane domains. The sequence also contained a mercurial-sensitive cysteine and a putative phosphorylation motif site for protein kinase A at Ser-257. The swelling assay using Xenopus oocytes revealed that AQP-x5 facilitated water permeability. Expression of AQP-x5 mRNA was restricted to the skin, brain, lungs and testes. Immunofluorescence and immunoelectron microscopical studies using an anti-peptide antibody (ST-156) against the C-terminal region of the AQP-x5 protein revealed the presence of immunopositive cells in the skin, with the label predominately localized in the apical plasma membrane of the secretory cells of the small granular glands. These glands are unique both in being close to the epidermal layer of the skin and in containing mitochondria-rich cells with vacuolar H+-ATPase dispersed among its secretory cells. Results from immunohistochemical experiments on the mucous or seromucous glands of several other anurans verified this result. We conclude that the presence of AQP-x5 in the apical plasma membrane of the small granular glands suggests its involvement in water secretion from the skins. The physiological roles of the AQP-x5 protein in the small or mucous glands are discussed. (+info)
A naturally occurring point mutation in the rat aquaporin 5 gene, influencing its protein production by and secretion of water from salivary glands.
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A greater than twofold diversity in the expression level of aquaporin 5 (AQP5) has been observed in the membrane fraction of the submandibular gland (SMG) in Sprague-Dawley rats (Murdiastuti K, Miki O, Yao C, Parvin MN, Kosugi-Tanaka C, Akamatsu T, Kanamori N, and Hosoi K. Pflugers Arch 445: 405-412, 2002). In the present study, breeding between brother and sister rats was repeated within high AQP5 producers and low ones to obtain inbred offspring. High- and low-producer rats from 3rd to 18th generations were used for experiments. By Western blotting, levels of AQP5 proteins in the parotid and lacrimal glands, and lungs were all low in low producers, whereas they were all high in high producers, implying genetic variations of the gene for this water channel. Despite this implication, AQP5 mRNA levels were almost the same between the two groups by Northern blotting, suggesting the irrelevance of transcriptional regulation for this diversity. AQP5 cDNAs from the SMGs of the two groups were sequenced. The nucleotide sequence of AQP5 cDNA from low producers indicated the existence of a point mutation at nt 308 (G308A), leading to a replacement of (103)Gly with (103)Asp in the third transmembrane domain, but no alteration was detected in the Kozak area. The existence of such a mutation was confirmed by the assessment of genomic DNA also. This mutation may have resulted in an abnormal membrane insertion or ineffective trafficking of AQP5, since the rats having this mutation showed extremely low membrane expression of AQP5 in the SMG acinar cells and decreased water secretion from their salivary glands. (+info)
Downregulation of aquaporins 1 and 5 in nasal gland by osmotic stress in ducklings, Anas platyrhynchos: implications for the production of hypertonic fluid.
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Using primers against highly conserved regions of mammalian and bird aquaporins in RT-PCR experiments, we amplified products derived from duck (Anas platyrhynchos) nasal gland RNA that were identified as homologues of mammalian and chicken aquaporin 1 and aquaporin 5 cDNAs by sequencing. Using digoxigenin-labelled probes derived from these PCR products in northern blot analyses of mRNA isolated from nasal glands of untreated (naive) or osmotically stressed ducklings (replacement of drinking water with a 1% NaCl solution), we observed a decrease in aquaporin 1 (AQP1) and aquaporin 5 (AQP5) mRNA abundance (by approximately 40%) during saline adaptation in the animals. Western blot analysis of AQP1 and AQP5 expression in the glands revealed that protein abundance decreased in a similar fashion. Immunohistochemical analysis of AQP1 distribution in cryosections of nasal gland indicated that AQP1 is mainly expressed in endothelial cells of the capillaries, but definitely not in the secretory or ductal cells of the gland. AQP5 distribution in the gland, however, seems to be different, since staining was exclusively observed in apical and basolateral plasma membranes of individual epithelial cells of the primary and central ducts, which collect fluid from the secretory tubules. The observations are consistent with the hypothesis that strongly hyperosmotic fluid is produced by the secretory cells at very low (unstimulated gland) or high (activated gland) rates. In the unstimulated gland, secretions may be diluted by aquaporin-mediated transcellular water flux while passing through the ductal system flushing the glandular ducts, thereby potentially preventing ascending infections. In the activated gland, however, downregulation of aquaporins in capillaries and duct cells may prevent dilution of the initially secreted fluid, enabling the animals to excrete large volumes of a highly concentrated salt solution. (+info)
Conditional deletion of the mouse Klf4 gene results in corneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells.
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The Kruppel-like transcription factor KLF4 is among the most highly expressed transcription factors in the mouse cornea (B. Norman, J. Davis, and J. Piatigorsky, Investig. Ophthalmol. Vis. Sci. 45:429-440, 2004). Here, we deleted the Klf4 gene selectively in the surface ectoderm-derived structures of the eye (cornea, conjunctiva, eyelids, and lens) by mating Klf4-LoxP mice (J. P. Katz, N. Perreault, B. G. Goldstein, C. S. Lee, P. A. Labosky, V. W. Yang, and K. H. Kaestner, Development 129:2619-2628, 2002) with Le-Cre mice (R. Ashery-Padan, T. Marquardt, X. Zhou, and P. Gruss, Genes Dev. 14:2701-2711, 2000). Klf4 conditional null (Klf4CN) embryos developed normally, and the adult mice were viable and fertile. Unlike the wild type, the Klf4CN cornea consisted of three to four epithelial cell layers; swollen, vacuolated basal epithelial and endothelial cells; and edematous stroma. The conjunctiva lacked goblet cells, and the anterior cortical lens was vacuolated in Klf4CN mice. Excessive cell sloughing resulted in fewer epithelial cell layers in spite of increased cell proliferation at the Klf4CN ocular surface. Expression of the keratin-12 and aquaporin-5 genes was downregulated, consistent with the Klf4CN corneal epithelial fragility and stromal edema, respectively. These observations provide new insights into the role of KLF4 in postnatal maturation and maintenance of the ocular surface and suggest that the Klf4CN mouse is a useful model for investigating ocular surface pathologies such as dry eye, Meesmann's dystrophy, and Steven's-Johnson syndrome. (+info)
Radioprotective effect of heat shock protein 25 on submandibular glands of rats.
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Irradiation (IR) is a fundamental treatment modality for head and neck malignancies. However, a significant drawback of IR treatment is irreversible damage of salivary gland in the IR field. In the present study, we investigated whether heat shock protein (HSP) 25 could be used as a radioprotective molecule for radiation-induced salivary gland damage in rats. HSP25 as well as inducible HSP70 (HSP70i) that were delivered to the salivary gland via an adenoviral vector significantly ameliorated radiation-induced salivary fluid loss. Radiation-induced apoptosis, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage in acinar cells, granular convoluted cells, and intercalated ductal cells were also inhibited by HSP25 or HSP70i transfer. The alteration of salivary contents, including amylase, protein, Ca+, Cl-, and Na+, was also attenuated by HSP25 transfer. Histological analysis revealed almost no radiation-induced damage in salivary gland when HSP25 was transferred. Aquaporin 5 expression in salivary gland was inhibited by radiation; and HSP25 transfer to salivary gland prevented this alteration. The protective effect of HSP70i on radiation-induced salivary gland damage was less or delayed than that of HSP25. These results indicate that HSP25 is a good candidate molecule to protect salivary gland from the toxicity of IR. (+info)