Aquaporin 5-deficient mouse lungs are hyperresponsive to cholinergic stimulation.
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Although aquaporin 5 (AQP5) is the major water channel expressed in alveolar type I cells in the lung, its actual role in the lung is a matter of considerable speculation. By using immunohistochemical staining, we show that AQP5 expression in mouse lung is not restricted to type I cells, but is also detected in alveolar type II cells, and in tracheal and bronchial epithelium. Aqp5 knockout (Aqp5(-/-)) mice were used to analyze AQP5 function in pulmonary physiology. Compared with Aqp5(+/+) mice, Aqp5(-/-) mice show a significantly increased concentration-dependent bronchoconstriction to intravenously administered Ach, as shown by an increase in total lung resistance and a decrease in dynamic lung compliance (P < 0.05). Likewise, Penh, a measure of bronchoconstriction, was significantly enhanced in Aqp5(-/-) mice challenged with aerosolized methacholine (P < 0.05). The hyperreactivity to bronchoconstriction observed in the Aqp5(-/-) mice was not due to differences in tracheal smooth muscle contractility in isolated preparations or to altered levels of surfactant protein B. These data suggest a novel pathway by which AQP5 influences bronchoconstriction. This observation is of special interest because studies to identify genetic loci involved in airway hyperresponsiveness associated with asthma bracket genetic intervals on human chromosome 12q and mouse chromosome 15, which contain the Aqp5 gene. (+info)
Bone marrow-derived cells as progenitors of lung alveolar epithelium.
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We assessed the capacity of plastic-adherent cultured bone marrow cells to serve as precursors of differentiated parenchymal cells of the lung. By intravenously delivering lacZ-labeled cells into wild-type recipient mice after bleomycin-induced lung injury, we detected marrow-derived cells engrafted in recipient lung parenchyma as cells with the morphological and molecular phenotype of type I pneumocytes of the alveolar epithelium. At no time after marrow cell injection, did we detect any engraftment as type II pneumocytes. In addition, we found that cultured and fresh aspirates of bone marrow cells can express the type I pneumocyte markers, T1alpha and aquaporin-5. These observations challenge the current belief that adult alveolar type I epithelial cells invariably arise from local precursor cells and raise the possibility of using injected marrow-derived cells for therapy of lung diseases characterized by extensive alveolar damage. (+info)
Functional requirement of aquaporin-5 in plasma membranes of sweat glands.
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The distribution and function of aquaporins (AQPs) have not previously been defined in sweat glands. In this study, AQP1, AQP3, and AQP5 mRNA were demonstrated in rat paw by reverse transcription (RT)-PCR, but AQP2 and AQP4 were not. AQP1, AQP3, and AQP5 protein were confirmed in these tissues by immunoblotting. AQP1 was identified in capillary endothelial cells by immunohistochemical labeling, but not in sweat glands or epidermis. Abundant AQP3 expression was seen in basal levels of epidermis, but not in sweat glands. AQP2 and AQP4 were not observed in either skin or sweat glands. Immunohistochemical labeling revealed abundant AQP5 in secretory parts of rat and mouse sweat glands, where immunoelectron microscopy demonstrated abundant AQP5 labeling in the apical plasma membrane. AQP5 immunolabeling of human sweat glands yielded a similar pattern. To establish the role of AQP5 in sweat secretion, we tested the response of adult mice to s.c. injection of pilocarpine, as visualized by reaction of secreted amylase with iodine/starch. The number of active sweat glands was dramatically reduced in AQP5-null (-/-) mice compared with heterozygous (+/-) and wild-type (+/+) mice. We conclude that the presence of AQP5 in plasma membranes of sweat glands is essential for secretion, providing potential insight into mechanisms underlying mammalian thermoregulation, tactile sensitivity, and the pathophysiology of hyperhidrosis. (+info)
Expression and localization of AQP5 in the stomach and duodenum of the rat.
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The expression, localization, and regulation of aquaporin 5 (AQP5), a member of the water channel family of proteins, was investigated in tissues of the rat gastrointestinal tract. Reverse transcriptase--polymerase chain reaction (RT--PCR) detected AQP5 mRNA in the lower stomach and duodenum. DNA sequencing confirmed that the cDNA fragment amplified had the complete sequence of the AQP5 cDNA fragment. Western blot analysis indicated the expression of a 27 kDa molecular mass AQP5 protein in the lower stomach and duodenum, which size was the same as that found for the protein in the submandibular gland and lungs. By immunohistochemistry using the IgG affinity-purified AQP5 antibody, the pyloric gland and Brunner's gland were primarily stained in the lower stomach and duodenum, respectively; a strong staining appeared in the apical and lateral membranes in both glands. These results indicate that AQP5 is present in the rat lower stomach and duodenum where it may be involved in a water transport mechanism. These results also support the idea that AQP5, and probably other aquaporins, are involved in water secretion in the stomach and duodenum although the volume of water transported via AQPs is unclear. (+info)
GATA6 regulates differentiation of distal lung epithelium.
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GATA6 is a member of the GATA family of zinc-finger transcriptional regulators and is the only known GATA factor expressed in the distal epithelium of the lung during development. To define the role that GATA6 plays during lung epithelial cell development, we expressed a GATA6-Engrailed dominant-negative fusion protein in the distal lung epithelium of transgenic mice. Transgenic embryos lacked detectable alveolar epithelial type 1 cells in the distal airway epithelium. These embryos also exhibited increased Foxp2 gene expression, suggesting a disruption in late alveolar epithelial differentiation. Alveolar epithelial type 2 cells, which are progenitors of alveolar epithelial type 1 cells, were correctly specified as shown by normal thyroid transcription factor 1 and surfactant protein A gene expression. However, attenuated endogenous surfactant protein C expression indicated that alveolar epithelial type 2 cell differentiation was perturbed in transgenic embryos. The number of proximal airway tubules is also reduced in these embryos, suggesting a role for GATA6 in regulating distal-proximal airway development. Finally, a functional role for GATA factor function in alveolar epithelial type 1 cell gene regulation is supported by the ability of GATA6 to trans-activate the mouse aquaporin-5 promoter. Together, these data implicate GATA6 as an important regulator of distal epithelial cell differentiation and proximal airway development in the mouse. (+info)
Expression of aquaporin-5 and its regulation in skeletal muscle cells.
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The aquaporins constitute a family of homologous intrinsic membrane proteins that function as highly selective water channels and are highly expressed in tissues where rapid water movement across the cell membrane is required. Molecular mechanism of water transport through the plasma membrane of skeletal muscle is still not clear. This study was designed to identify aquaporin subtypes and their expression regulation in C2C12 cells, a mouse myoblastic cell line. RT-PCR, immunohistochemistry and Western blot analysis revealed that C2C12 cells express AQP5. AQP5 expression was increased by induction of C2C12 differentiation. Exposure of C2C12 cells to hypertonic solutions induced an increase in AQP5 expression and p38 kinase activation. However, a p38 kinase inhibitor failed to inhibit hyperosmolar induction of AQP5 expression in C2C12 cells. These data indicate that skeletal muscle cells express AQP5 protein and its expression is regulated by differentiation and hypertonic stress. (+info)
The muscarinic acetylcholine receptor-stimulated increase in aquaporin-5 levels in the apical plasma membrane in rat parotid acinar cells is coupled with activation of nitric oxide/cGMP signal transduction.
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The present study investigated the role of nitric oxide (NO)/cGMP signal transduction in the M(3) muscarinic acetylcholine receptor (mAChR)-stimulated increase in aquaporin-5 (AQP5) levels in the apical plasma membrane (APM) of rat parotid glands. Pretreatment of rat parotid tissue with the NO scavenger 2-(4carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium inhibited both acetylcholine (ACh)- and pilocarpine-induced increases in AQP5 in the APM. NO donors [3-morpholinosydnonimine (SIN-1) and (S)-nitroso-N-acetylpenicillamine (SNAP)] mimicked the effects of mAChR agonists. A selective protein kinase G inhibitor [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1 H-diindolo-[1,2,3-fg-3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT5823)] and an NO synthase inhibitor (N(6)-imminoethyl-L-lysine) blocked SIN-1- and SNAP-induced increases in AQP5 in the APM. A calmodulin kinase II inhibitor [(8)-5-isoquinolinesulfonic acid, 4-[2-(5-isoquinolinyl-sulfonyl)methylamino]-3-oxo-(4-phenyl-1-piperazinyl)-propyl ]phenyl ester (KN-62)] decreased the pilocarpine-induced increase of AQP5 in the APM. Using diaminofluorescinein-2 diacetate, enhanced NO synthase activity was detected in isolated parotid acinar cells after ACh-treatment. Treatment with dibutyryl cGMP, but not dibutyryl cAMP, induced an increase in AQP5 levels in the APM. BAPTA-AM inhibited the cGMP-induced increase in AQP5 in the APM. Pretreatment of the tissues with a myosin light chain kinase inhibitor [(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9)] inhibited a mAChR-stimulated increase in AQP5 levels in the APM. Although there was a significant ACh-induced increase in AQP5 in the APM in the absence of extracellular Ca(2+), the maximal effect of ACh on the AQP5 levels in the APM occurred in the presence of extracellular Ca(2+). These results suggest that NO/cGMP signal transduction has a crucial role in Ca(2+) homeostasis in the mAChR-stimulated increase in AQP5 levels in the APM of rat parotid glands. (+info)
Localization of aquaporin-5 in sweat glands and functional analysis using knockout mice.
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Sweat secretion involves the transport of salt and water into the lumen of the secretory coil of the sweat gland. By analogy to salivary and submucosal glands, where fluid secretion is aquaporin-5 (AQP5) dependent, we postulated that aquaporin water channels might facilitate sweat secretion. Immunolocalization with specific antibodies revealed strong expression of AQP5 at the luminal membrane of secretory epithelial cells in sweat glands in mouse paw skin. Novel quantitative methods were developed to compare sweat secretion in wild-type mice and mice lacking AQP5. Total hindpaw sweat secretion was measured by proton nuclear magnetic resonance of sweat-derived (1)H(2)O in (2)H(2)O solvent, and sweat secretion from individual glands was measured by real-time video imaging of sweat droplet formation under oil. Sweat secretion rates after pilocarpine stimulation did not differ in wild-type mice (0.21 +/- 0.03 nl min(-1) gland(-1)) vs. mice lacking AQP5 (0.19 +/- 0.04 nl min(-1) gland(-1)). The lack of effect of AQP5 on sweat secretion rate was confirmed by microcapillary collections of sweat from defined regions of mouse paws. Also, as by direct counting of droplets, the number of functional sweat glands was not affected by AQP5 deletion. Sweat gland morphology was similar in wild-type and AQP5 null mice. From sweat coil geometry and gland secretion rate, the rate of fluid secretion was estimated to be 130 nl min(-1) cm(-2) of secretory epithelium, substantially lower than that of > 500 nl min(-1) cm(-2) in kidney proximal tubules and salivary glands, where active fluid absorption or secretion is aquaporin dependent. These results indicate the expression of AQP5 in sweat gland secretory epithelium, but provide direct evidence against its physiological involvement in sweat fluid secretion in mice. (+info)