Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The aquaporins transport water through membranes of numerous tissues, but the molecular mechanisms for sensing changes in extracellular osmolality and regulating water balance in brain are unknown. We have isolated a brain aquaporin by homology cloning. Like aquaporin 1 (AQP1, also known as CHIP, channel-forming integral membrane protein of 28 kDa), the deduced polypeptide has six putative transmembrane domains but lacks cysteines at the known mercury-sensitive sites. Two initiation sites were identified encoding polypeptides of 301 and 323 amino acids; expression of each in Xenopus oocytes conferred a 20-fold increase in osmotic water permeability not blocked by 1 mM HgCl2, even after substitution of cysteine at the predicted mercury-sensitive site. Northern analysis and RNase protection demonstrated the mRNA to be abundant in mature rat brain but only weakly detectable in eye, kidney, intestine, and lung. In situ hybridization of brain localized the mRNA to ependymal cells lining the aqueduct, glial cells forming the edge of the cerebral cortex and brainstem, vasopressin-secretory neurons in supraoptic and paraventricular nuclei of hypothalamus, and Purkinje cells of cerebellum. Its distinctive expression pattern implicates this fourth mammalian member of the aquaporin water channel family (designated gene symbol, AQP4) as the osmoreceptor which regulates body water balance and mediates water flow within the central nervous system.
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PMID:Molecular characterization of an aquaporin cDNA from brain: candidate osmoreceptor and regulator of water balance. 752 31

The RNase protection assay was applied to quantify mRNA expression of five principal mammalian water channels in 18 different rat tissues, and to determine the influence of dehydration on renal water channel expression. Probes consisted of labeled cRNAs transcribed from cDNA fragments of rat CHIP28 (AQP-1, bp 238-575 of coding sequence), AQP-CD (AQP2, bp 53-606), MIWC (AQP4, bp 235-572), GLIP (AQP3, bp 219-604), and AQP5 (bp 56-612). Results were normalized to expression of rat beta-actin by quantitative densitometry of autoradiograms. CHIP28 mRNA was expressed strongly in heart, kidney > placenta, skeletal muscle, and urinary bladder and detected weakly in eye, lung, trachea, spleen, liver, colon, prostate, and skin. AQP-CD was detected only in kidney. MIWC mRNA expression was highest in brain, followed by eye, trachea, lung, stomach, kidney, and skeletal muscle. GLIP was found in eye, trachea, kidney, urinary bladder, skin, prostate, placenta, and skeletal muscle. AQP5 was detected in salivary gland, eye, lung, and trachea. An alternatively spliced form of MIWC (sMIWC) was also identified in lung and kidney by RNase protection assay, corresponding to deletion of exon 2 of MIWC. In response to dehydration (3 days, -15 % body weight), renal expression of CHIP28 and MIWC were unchanged, whereas expression of AQP-CD and GLIP were increased significantly by 2.18 +/- 0.04 and 1.36 +/- 0.11 fold (SE, n = 5), respectively. These results establish quantitative values for aquaporin transcript expression in multiple mammalian tissues. The sensitive RNase protection assay revealed the expression of water channels in several tissues not studied previously or in which mRNA levels were too low to detect by Northern blot analysis. The observation of GLIP up-regulation in kidney by dehydration suggests a role in the urinary concentrating mechanism.
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PMID:Quantitative analysis of aquaporin mRNA expression in rat tissues by RNase protection assay. 867 43

The aquaporin family of membrane water transport proteins are expressed in diverse tissues, and in brain the predominant water channel protein is AQP4. Here we report the isolation and characterization of the human AQP4 cDNAs and genomic DNA. Two cDNAs were isolated corresponding to the two initiating methionines (M1 in a 323-aa polypeptide and M23 in a 301-aa polypeptide) previously identified in rat [Jung, J.S., Bhat, R.V., Preston, G.M., Guggino, W.B. & Agre, P. (1994) Proc. Natl. Acad. Sci. USA 91, 13052-13056]. Similar to other aquaporins, the AQP4 gene is composed of four exons encoding 127, 55, 27, and 92 amino acids separated by introns of 0.8, 0.3, and 5.2 kb. Unlike other aquaporins, an alternative coding initiation sequence (designated exon 0) was located 2.7 kb upstream of exon 1. When spliced together, M1 and the subsequent 10 amino acids are encoded by exon 0; the next 11 amino acids and M23 are encoded by exon 1. Transcription initiation sites have been mapped in the proximal promoters of exons 0 and 1. RNase protection revealed distinct transcripts corresponding to M1 and M23 mRNAs, and AQP4 immunoblots of cerebellum demonstrated reactive polypeptides of 31 and 34 kDa. Using a P1 and a lambda EMBL subclone, the chromosomal site of the human AQP4 gene was mapped to chromosome 18 at the junction of q11.2 and q12.1 by fluorescence in situ hybridization. These studies may now permit molecular characterization of AQP4 during human development and in clinical disorders.
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PMID:The human AQP4 gene: definition of the locus encoding two water channel polypeptides in brain. 885 81

Three members of the water channel (aquaporin) family are expressed in adult rat lung: CHIP28 (AQP-1), MIWC (AQP-4), and AQP-5. Because water channels may be important in the clearance of fluid from the newborn lung, the expression of water channels just before and after birth was investigated using the ribonuclease (RNAse) protection assay. RNA was isolated from lungs, brain, and heart of prenatal rats (fetal days F19, F20, and F21) and postnatal rats (days +1, +2, +5, +7, +21, and adult). Transcript expression was measured relative to a beta-actin control by quantitative densitometry. Whereas beta-actin mRNA expression was nearly constant over time, distinct expression patterns were observed for the three water channels. CHIP28 mRNA expression rose slowly from days F19 to +1, then strongly at day +2, and remained elevated over the first week. MIWC mRNA was weakly expressed prenatally, but strongly increased just after birth. AQP-5 mRNA increased slowly and monotonically between days F20 and +7. These patterns contrasted sharply with the developmental expression of CHIP28 in heart, which decreased over time, and MIWC in brain. Immunocytochemistry showed CHIP28 protein expression in capillary endothelia and MIWC in airway epithelia by day +1; quantitative immunoblot analysis showed increased CHIP28 protein expression over time. These findings are consistent with a role of lung water channels in perinatal fluid clearance; however, proof of physiologic significance will require functional measurements of air space-capillary water permeability.
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PMID:Sharp increase in rat lung water channel expression in the perinatal period. 891 74

Aquaporin-2 (AQP-2) water channel is a key molecule for urinary concentration whose expression is augmented by dehydration in vivo. To elucidate the regulatory mechanism of this phenomenon in vitro, mouse collecting duct cell lines were established from a transgenic mouse harboring temperature-sensitive simian virus 40 large T antigen gene and then screened for the AQP-2 expression, using ribonuclease protection assay. In one cell line designated C4, the endogenous AQP-2 mRNA level measured by ribonuclease protection assay increased fourfold after treatment with chlorophenylthio-cAMP (cpt-cAMP) (400 microM). In contrast, phorbol 12-myristate 13-acetate did not affect the AQP-2 mRNA level. To identify the molecular mechanism(s) of cAMP-induced upregulation of AQP-2 mRNA in C4 cells, luciferase assay was performed using various 5'-flanking regions of the human AQP-2 gene. Luciferase activity in C4 cells transfected with constructs containing approximately 2.8-kbp or 224-bp 5'-flanking region showed a 3.5-fold increase by cpt-cAMP treatment, indicating that the 224-bp 5'-flanking region contains the elements necessary for cAMP-induced regulatory mechanisms. This region contains cAMP-responsive element (CRE), and the deletion of the core sequence of CRE (GACGTCA) or introduction of mutation into CRE (GTGGTCA) completely abolished the responsiveness to cpt-cAMP, confirming the key role of CRE in the cAMP-induced transcriptional activation of the AQP-2 gene. Electrophoretic mobility shift assay revealed the existence of proteins binding to CRE in C4 cells and in rat kidney. The binding of CRE proteins to CRE was increased in the nuclear extract from cpt-cAMP-treated C4 cells and dehydrated rat kidney compared with those from controls. These results demonstrated that the CRE in the AQP-2 gene promoter is a key cis-element for cAMP-mediated transcriptional regulation of this gene and may be important for in vivo regulation of AQP-2 expression in a dehydrated state.
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PMID:Transcriptional regulation of aquaporin-2 water channel gene by cAMP. 918 51

The gene encoding the mouse Aquaporin-8 water channel protein (Aqp8) was cloned and its genomic structure was defined. Aqp8 consists of six exons with boundaries at amino acids 1-4, 5-87, 88-129, 130-201, 202-246 and 247-261 which partially correspond to those of other known aquaporin genes. All splice sites conform to the GT-AG rule except the first one which is GT-GG. Primer extension and RNase protection analyses using mouse liver RNA demonstrated three initiation transcription sites located 385, 156 and 146 bp upstream from the translational start codon. No defined TATA box was found in the 5'-flanking region where numerous CAAT motifs and one GATA box were identified. Fluorescence in situ hybridization localized the Aqp8 locus to mouse chromosome 7F3. The 7F region is syntenic with human chromosomes 11, 16 and 10. These results (i) reveal marked structural distinction between the Aqp8 gene and the other known mammalian aquaporin genes, (ii) may now permit the molecular characterization of Aqp8 expression and (iii) represent a fundamental step for the construction of a target vector to generate transgenic Aqp8 knockout mice.
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PMID:Cloning, structural organization and chromosomal localization of the mouse aquaporin-8 water channel gene (Aqp8). 1044 5

We recently reported that secretin induces the exocytic insertion of functional aquaporin-1 water channels (AQP1) into the apical membrane of cholangiocytes and proposed that this was a key process in ductal bile secretion. Because AQP1 is present on the basolateral cholangiocyte membrane in low amounts, we hypothesized that another AQP must be expressed at this domain to facilitate transbasolateral water movement. Thus, we investigated the expression, subcellular localization, possible regulation by secretin, and functional activity of AQP4, a mercury-insensitive water channel expressed in other fluid transporting epithelia. Using reverse transcription-polymerase chain reaction (RT-PCR) on RNA prepared from purified rat cholangiocytes, we amplified a product of 311 bp that was 100% homologous to the reported AQP4 sequence. RNase protection assay confirmed the presence of an appropriate size transcript for AQP4 in cholangiocytes. Immunoblotting detected a band of approximately 31 kd corresponding to AQP4 in basolateral but not apical membranes of cholangiocytes. Secretin did not alter the amount of plasma membrane AQP4 but, as expected, induced AQP1 redistribution from intracellular to apical plasma membranes. Functional studies showed that AQP4 accounts for about 15% of total cholangiocyte membrane water permeability. Our results indicate that: (1) cholangiocytes express AQP4 messenger RNA (mRNA) and protein and (2) in contrast to AQP1, which is targeted to the apical cholangiocyte membrane by secretin, AQP4 is constitutively expressed on the basolateral cholangiocyte membrane and is secretin unresponsive. The data suggest that AQP4 facilitates the basolateral transport of water in cholangiocytes, a process that could be relevant to ductal bile formation.
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PMID:Expression of aquaporin-4 water channels in rat cholangiocytes. 1082 57

The aquaporin (AQP)-9 gene was recently isolated from human and rat liver cDNA libraries as a member of the water channel family for water and neutral solutes. Although the expression of AQP9 mRNA has been demonstrated in several organs including the liver and testis by Northern blot analysis, the cellular and subcellular localization of the AQP9 protein remains unclear. In the present light and electron microscopic immunohistochemical study, the localization of the AQP9 immunoreactivity was examined in fifteen kinds of rat organs using an antibody against rat AQP9 synthetic peptide. The antibody immunostained a major band of approximately 33 kDa in the liver by Western blot analysis. Immunoreactivity for AQP9 was found exclusively in the liver and testis among the organs examined. In the liver, positive staining appeared selectively along the space of Disse. Immunoelectron microscopy confirmed the localization of AQP9 on the surface of hepatocyte microvilli facing the space of Disse. In the testis, the plasma membrane of Leydig cells located between seminiferous tubules was conspicuously immunoreactive to the antibody. Intense mRNA expression was detected in the liver and testis but not in other organs by ribonuclease protection assay. These findings suggest a specific role for AQP9 in the transport of water and non-charged solutes in hepatocytes and Leydig cells.
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PMID:Immunolocalization of aquaporin-9 in rat hepatocytes and Leydig cells. 1131 May 8

Expression of aquaporin-8 mRNA has previously been shown in hepatocytes, pancreatic acinar cells, colon epithelial cells and seminiferous tubules of the testis in the rat by in situ hybridization technique. However, immunolocalization of this water channel has not yet been demonstrated. In the present study, the localization of immunoreactive aquaporin-8 and expression of the mRNA were examined in rat organs (cerebrum, cerebellum, eye, salivary gland, heart, lung, liver, pancreas, esophagus, stomach, jejunum, ileum, colon, testis, ovary, kidney, spleen and lymphnode) by immunohistochemistry using an antibody against aquaporin-8 and ribonuclease protection assay. Aquaporin-8 was distinctly immunolocalized on the apical membranes of pancreatic acinar cells and mucosal epithelium of the colon and jejunum. In the liver, the bile canalicular membrane of hepatocytes was immunostained. In the testis, immunoreactive aquaporin-8 was demonstrated on the luminal side of the seminiferous tubules. At high magnification, the peroxidase reaction products appeared on the ramified cytoplasmic membrane of Sertoli cells surrounding the residual bodies or spermatogenic cells. Specificity of the antibody was verified by Western blot analysis showing a minor approximately 28 kDa band (deduced deglycosylated form of aquaporin-8) and a major approximately 30 kDa band (glycosylated form) in these organs. The intensity of aquaporin-8 immunoreactivity was approximately comparable to that of aquaporin-8 mRNA expression in the liver, pancreas, colon, jejunum and testis. The aquaporin-8 mRNA expression in the hepatocytes was presumed to be closely associated with the structure of bile canaliculi since the message was detected in hepatocytes immediately after isolation from the liver but not in cells following cultivation for three days. The localization of immunoreactive aquaporin-8 indicated functions for this water channel in the secretion of bile and pancreatic juice, and the secretion or absorption of water in the colon and jejunum, and the maturation or liberation of spermatogenic cells in the testis.
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PMID:Immunolocalization of aquaporin-8 in rat digestive organs and testis. 1143 86

Fine regulation of water reabsorption by the antidiuretic hormone [8-arginine]vasopressin (AVP) occurs in principal cells of the collecting duct and is largely dependent on regulation of the aquaporin-2 (AQP2) water channel. AVP-inducible long term AQP2 expression was investigated in immortalized mouse cortical collecting duct principal cells. Combined RNase protection assay, Western blot, and immunofluorescence analyses revealed that physiological concentrations of AVP added to the basal side, but not to the apical side, of cells grown on filters induced both AQP2 mRNA and apical protein expression. The stimulatory effect of AVP on AQP2 expression followed a V(2) receptor-dependent pathway because [deamino-8-d-arginine]vasopressin (dDAVP), a specific V(2) receptor agonist, produced the same effect as AVP, whereas the V(2) antagonist SR121463B antagonized action of both AVP and dDAVP. Moreover, forskolin and cyclic 8-bromo-AMP fully reproduced the effects of AVP on AQP2 expression. Analysis of protein degradation pathways showed that inhibition of proteasomal activity prevented synthesis of AVP-inducible AQP2 mRNA and protein. Once synthesized, AQP2 protein was quickly degraded, a process that involves both the proteasomal and lysosomal pathways. This is the first study that delineates induction and degradation mechanisms of AQP2 endogenously expressed by a renal collecting duct principal cell line.
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PMID:Long term regulation of aquaporin-2 expression in vasopressin-responsive renal collecting duct principal cells. 1178 89


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