Search PubMed⌕ Search

Biomedical subjects

P Agre

Publications and source records attributed to P Agre.

At least 19 recordsLinked to original sources
PubMed1996

The human AQP4 gene: definition of the locus encoding two water channel polypeptides in brain.

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.

Alternative Splicing↗
PubMed1996

Adenovirus-mediated expression of aquaporin-5 in epithelial cells.

A recombinant adenovirus coding for rat aquaporin-5 was constructed and plaque purified. The recombinant adenovirus (AdrAQP5) mediated the expression of aquaporin-5 in rat and human salivary cell lines and in dog kidney cells in vitro as demonstrated by Northern blot and Western blot analyses, and by confocal microscopy after immunofluorescent labeling. In kidney cells, expression of the transgene was optimal if cells were infected at their basolateral surface, a phenomenon associated with the distribution of integrin receptors on these cells. The expressed aquaporin-5 protein was functionally active because viral-mediated gene transfer resulted in a significant increase in the osmotically directed net fluid secretion rate across monolayers of kidney cells. AdrAQP5 should provide an efficient and useful means to impart facilitated water permeability to cells lacking such a pathway.

Adenoviridae↗
PubMed1996

Aquaporin-1 water channel protein in lung: ontogeny, steroid-induced expression, and distribution in rat.

At birth water is rapidly reabsorbed from the distal lung in preparation for alveolar gas exchange. To investigate a potential role for the AQP1 water channel in development, lung membranes from fetal and perinatal rats were analyzed by immunoblot. First expression of AQP1 was noted in fetal rat lung at E19 (19th day of the 21-day gestation). The level of AQP1 increased fivefold from the last gestational day to the first postnatal day and persisted at high levels into adulthood. Maternal corticosteroids increased expression of AQP1 in fetal lung, an effect also seen in adult rats. AQP1 mRNA increased in rat pups treated with corticosteroids, suggesting at least partial regulation at the level of transcription. Immunohistochemical analyses with anti-AQP1 demonstrated the protein in peribronchial vessels and visceral pleura at E21 with increased postnatal expression. AQP1 was not expressed in airway epithelium, and only occasional alveolar pneumocytes were labeled. Immunoelectron microscopy revealed AQP1 on both apical and basolateral membranes of endothelial cells. The ontogeny and corticosteroid induction of AQP1 in rat lung coincide with major physiological alterations in lung development; however, the distribution of AQP1 predicts the existence of other water channels in the alveolar epithelium.

Adrenal Cortex Hormones↗
PubMed1996

The human Aquaporin-5 gene. Molecular characterization and chromosomal localization.

The cDNA for the fifth mammalian aquaporin (AQP5) was isolated from rat, and expression was demonstrated in rat salivary and lacrimal glands, cornea, and lung (Raina, S., Preston, G. M., Guggino, W. B., and Agre, P. (1995) J. Biol. Chem. 270, 1908-1912). Here we report the isolation and characterization of the human AQP5 cDNA and gene. The AQP5 cDNA from a human submaxillary gland library contains a 795-base pair open reading frame encoding a 265-amino acid protein. The deduced amino acid sequences of human and rat AQP5 are 91% identical with 6 substitutions in the 22-amino acid COOH-terminal domain. Expression of human AQP5 in Xenopus oocytes conferred mercurial-sensitive osmotic water permeability (Pf) equivalent to other aquaporins. The human AQP5 structural gene resides within a 7. 4-kilobase SalI-EcoRI fragment with four exons corresponding to amino acids 1-121, 122-176, 177-204, and 205-265 separated by introns of 1.2, 0.5, and 0.9 kilobases. A transcription initiation site was identified 518 base pairs upstream of the initiating methionine. Genomic Southern analysis indicated that AQP5 is a single copy gene which localized to human chromosome 12q13; this coincides with the chromosomal locations of the homologous human genes MIP and AQP2, thus confirming 12q13 as the site of an aquaporin gene cluster. The mouse gene localized to distal chromosome 15. This information may permit molecular characterization of AQP5 expression during normal development and in clinical disorders.

Amino Acid Sequence↗
PubMed1996

Functional analysis of aquaporin-1 deficient red cells. The Colton-null phenotype.

The aquaporin-1 (AQP1) water transport protein contains a polymorphism corresponding to the Colton red blood cell antigens. To define the fraction of membrane water permeability mediated by AQP1, red cells were obtained from human kindreds with the rare Colton-null phenotype. Homozygosity or heterozygosity for deletion of exon I in AQP1 correlated with total or partial deficiency of AQP1 protein. Homozygote red cell morphology appeared normal, but clinical laboratory studies revealed slightly reduced red cell life span in vivo; deformability studies revealed a slight reduction in membrane surface area. Diffusional water permeability (Pd) was measured under isotonic conditions by pulsed field gradient NMR. Osmotic water permeability (Pf) was measured by change in light scattering after rapid exposure of red cells to increased extracellular osmolality. AQP1 contributes approximately 64% (Pd = 1.5 x 10(-3) cm/s) of the total diffusional water permeability pathway, and lipid permeation apparently comprises approximately 23%. In contrast, AQP1 contributes > 85% (Pf = 19 x 10(-3) cm/s) of the total osmotic water permeability pathway, and lipid permeation apparently comprises only approximately 10%. The ratio of AQP1-mediated Pf to Pd predicts the length of the aqueous pore to be 36 A.

Aquaporin 1↗
PubMed1996

The aquaporin family of water channels in kidney: an update on physiology and pathophysiology of aquaporin-2.

The long-standing problem of membrane water transport has been advanced by the recognition of a new family of water transport proteins, the "aquaporins" [1-3]. Not surprisingly, water transport is a major process in kidney physiology, and the biology of aquaporins is most thoroughly understood in that organ. We reviewed in detail the status of aquaporins in the kidney only one year ago [4], but the subsequent progress has dictated the need for an update. This seems especially appropriate in honor of the 100th birthday of Homer Smith, the pioneer whose foresight initiated this field.

Animals↗
PubMed1996

Pathophysiology of the aquaporin water channels.

Discovery of aquaporin water channel proteins has provided insight into the molecular mechanism of membrane water permeability. The distribution of known mammalian aquaporins predicts roles in physiology and disease. Aquaporin-1 mediates proximal tubule fluid reabsorption, secretion of aqueous humor and cerebrospinal fluid, and lung water homeostasis. Aquaporin-2 mediates vasopressin-dependent renal collecting duct water permeability; mutations or downregulation can cause nephrogenic diabetes insipidus. Aquaporin-3 in the basolateral membrane of the collecting duct provides an exit pathway for reabsorbed water. Aquaporin-4 is abundant in brain and probably participates in reabsorption of cerebrospinal fluid, osmoregulation, and regulation of brain edema. Aquaporin-5 mediates fluid secretion in salivary and lacrimal glands and is abundant in alveolar epithelium of the lung. Specific regulation of membrane water permeability will likely prove important to understanding edema formation and fluid balance in both normal physiology and disease.

Animals↗
PubMed1996

Expression of aquaporins-1 and -2 during nephrogenesis and in autosomal dominant polycystic kidney disease.

Aquaporin-1 (AQP1), located in proximal tubules (PT) and descending thin limbs of Henle (DTL), and aquaporin-2 (AQP2), located in collecting ducts (CD), are channels involved in water transport across renal tubule epithelia. Using antibodies against AQP1 and AQP2, we here show expression of AQP1 and AQP2 in normal human developing and adult kidneys and in autosomal dominant polycystic kidney disease (ADPKD). Unlike in rats, AQP1 and AQP2 are expressed early during human nephrogenesis (12-wk gestation). AQP1 was first seen in developing PT epithelia, predominantly in apical cell membranes, and, at 15 wk, was also detected in DTL. AQP2 was seen in apical cell membranes of the branching ureteric bud and CD system from 12 wk and throughout development. In adult normal kidneys, AQP1 was localized to apical and basolateral membrane domains of PT and DTL, whereas AQP2 was restricted to principal cells of CD. This distribution of AQP1 and AQP2 was also seen in early stage ADPKD, except that AQP1 was mostly located in the apical membrane region of expanded PT. In end-stage ADPKD, two-thirds of the cysts expressed either AQP1 or AQP2, but these two water channels were never colocalized in the same cyst. Western blot analysis showed maximal expression of AQP1 and AQP2 in normal adult kidneys, lower levels in fetal kidneys, and decreases associated with degree of cystic progression in ADPKD. These data 1) demonstrate specific, mutually exclusive localization of AQP1 and AQP2 in human fetal and adult kidneys; 2) show that both channels are expressed early during nephrogenesis; and 3) show that the mutual exclusivity of localization is maintained even into end-stage ADPKD.

Aging↗
PubMed1995

Molecular cloning and characterization of AqpZ, a water channel from Escherichia coli.

The aquaporin family of molecular water channels is widely expressed throughout the plant and animal kingdoms. No bacterial aquaporins are known; however, sequence-related bacterial genes have been identified that encode glycerol facilitators (glpF). By homology cloning, a novel aquaporin-related DNA (aqpZ) was identified that contained no surface N-glycosylation consensus. The aqpZ RNA was not identified in mammalian mRNA by Northern analysis and exhibited bacterial codon usage preferences. Southern analysis failed to demonstrate aqpZ in mammalian genomic DNA, whereas a strongly reactive DNA was present in chromosomal DNA from Escherichia coli and other bacterial species and did not correspond to glpF. The aqpZ DNA isolated from E. coli contained a 693-base pair open reading frame encoding a polypeptide 28-38% identical to known aquaporins. When compared with other aquaporins, aqpZ encodes a 10-residue insert preceding exofacial loop C, truncated NH2 and COOH termini, and no cysteines at known mercury-sensitive sites. Expression of aqpZ cRNA conferred Xenopus oocytes with a 15-fold increase in osmotic water permeability, which was maximal after 5 days of expression, was not inhibited with HgCl2, exhibited a low activation energy (Ea = 3.8 kcal/mol), and failed to transport nonionic solutes such as urea and glycerol. In contrast, oocytes expressing glpF transported glycerol but exhibited limited osmotic water permeability. Phylogenetic comparison of aquaporins and homologs revealed a large separation between aqpZ and glpF, consistent with an ancient gene divergence.

Amino Acid Sequence↗
PubMed1995

The mouse aquaporin-1 gene.

Members of the aquaporin family of molecular water transporters are expressed in diverse epithelia and in complex developmental patterns. Using a cDNA for mouse Aqp1, the structural gene was isolated and a restriction map was constructed. The 13-kb Aqp1 gene contains four exons with intronic boundaries corresponding to other known aquaporin genes. Transcription begins 67 bp 5' to the translation initiation site and 20 bp 3' from a TATAA consensus sequence. Aqp1 was localized by interspecific mouse backcross mapping to the central region of mouse chromosome 6 syntenic with human chromosome 7p14, where AQP1 had previously been localized. These studies have revealed marked structural similarities between the mouse Aqp1 and the human AQP1 genes, suggesting that further comparative studies may provide molecular insight into genetic regulatory features shared by both species.

Animals↗
PubMed1995

Water channel properties of major intrinsic protein of lens.

The functions of major intrinsic protein (MIP) of lens are still unresolved; however the sequence homology with channel-forming integral membrane protein (CHIP) and other Aquaporins suggests that MIP is a water channel. Immunolocalizations confirmed that Xenopus oocytes injected with bovine MIP cRNA express the protein and target it to the plasma membrane. Control oocytes or oocytes expressing MIP or CHIP exhibited small, equivalent membrane currents that could be reversibly increased by osmotic swelling. When compared with water-injected control oocytes, the coefficient of osmotic water permeability (Pf) of MIP oocytes was increased 4-5-fold with a low Arrhenius activation energy, while the Pf of CHIP oocytes increased > 30-fold. To identify structures responsible for these differences in Pf, recombinant MIP proteins were expressed. Analysis of MIP-CHIP chimeric proteins revealed that the 4-kDa cytoplasmic domain of MIP did not behave as a negative regulator. Individual residues in MIP were replaced by residues conserved among the Aquaporins, and introduction of a proline in the 5th transmembrane domain of MIP raised the Pf by 50%. Thus oocytes expressing MIP failed to exhibit ion channel activity and consistently exhibited water transport by a facilitated pathway that was qualitatively similar to the Aquaporins but of lesser magnitude. We conclude that MIP functions as an Aquaporin in lens, but the protein may also have other essential functions.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and characterization of an aquaporin cDNA from salivary, lacrimal, and respiratory tissues.

The Aquaporin family of water channels plays a fundamental role in transmembrane water movements in numerous plant and animal tissues. Since the molecular pathway by which water is secreted by salivary glands is unknown, a cDNA was isolated from rat submandibular gland by homology cloning. Similar to other Aquaporins, the salivary cDNA encodes a 265-residue polypeptide with six putative transmembrane domains separated by five connecting loops (A-E); the NH2- and COOH-terminal halves of the polypeptide are sequence-related, and each contains the motif Asn-Pro-Ala. A mercurial-inhibition site is present in extracellular loop E, and cytoplasmic loop D contains a cAMP-protein kinase phosphorylation consensus. In vitro translation yielded a 27-kDa polypeptide, and expression of the cRNA in Xenopus oocytes conferred a 20-fold increase in osmotic water permeability (Pf) which was reversibly inhibited by 1 mM HgCl2. Northern analysis demonstrated a 1.6-kilobase mRNA in submandibular, parotid, and sublingual salivary glands, lacrimal gland, eye, trachea, and lung. In situ hybridization revealed a strong hybridization over the corneal epithelium in eye and over the secretory lobules in salivary glands. These studies have identified a new mammalian member of the Aquaporin water channel family (gene symbol AQP5) which is implicated in the generation of saliva, tears, and pulmonary secretions.

Amino Acid Sequence↗
PubMed1995

Aquaporin water channels: unanswered questions and unresolved controversies.

The long-standing biophysical question of how water crosses plasma membranes has been answered by the recent discovery of the aquaporins. Identification of this large family of membrane water-transport proteins has generated new questions about the physiological functions, tissue distributions, and regulatory mechanisms of individual aquaporins. The fast pace of developments in this field has also resulted in major discrepancies in published reports which warrant resolution.

Animals↗
PubMed1995

ABH and Colton blood group antigens on aquaporin-1, the human red cell water channel protein.

The recent identification of the red cell water transporter (AQP1) has led to the identification of the "aquaporins", a new class of membrane proteins which function as water-selective transport proteins and are involved in many physiological processes. Identification of the chromosomal localization of the corresponding gene led to the recognition that AQP1 is the structural basis of the Colton blood group antigens. Analysis of individuals with the Colton null phenotype led to the recognition that homozygosity for knockout mutations in the corresponding gene is exceedingly rare but is without a significant clinical phenotype, predicting a redundancy in expression of other aquaporin homologs. These studies demonstrate the importance which molecular studies in red cell blood group antigens may play in diverse areas of biomedical research. Moreover, they provide another example that blood group antigens may be polymorphisms in functionally important proteins on the red cell surface.

ABO Blood-Group System↗
PubMed1995

The aquaporin family of water channels in kidney.

The longstanding puzzle of membrane water permeability was advanced by the discovery of channel-forming integral protein (CHIP). This protein was shown to function as a water channel when expressed in Xenopus oocytes or when reconstituted into synthetic membranes. Site-directed mutagenesis and electron crystallography reveal tetrameric organization of CHIP, and the two halves of CHIP are tandem repeats folded into an obversely symmetric structure which resembles an hourglass. Each tetramer is comprised of functionally independent subunits. CHIP is the archetypal member of a newly-recognized family of membrane water transporters known as the "Aquaporins" (AQPs). AQP1 (CHIP) is abundant in the apical and basolateral membranes of renal proximal tubules and descending thin limbs, and is also present in a number of extra renal tissues. In the collecting duct, AQP2 is the predominant vasopressin-sensitive water channel. AQP2 is localized in the apical membrane and in intracellular vesicles which are targeted to the apical plasma membranes when stimulated by antidiuretic hormone. Humans are identified with mutations in AQP1 and AQP2 and exhibit contrasting clinical phenotypes. AQP3 resides in the basolateral membranes of collecting duct principal cells providing an exit pathway for water, and AQP4 is abundant in brain, where it apparently functions as the hypothalamic osmoreceptor responsible for secretion of antidiuretic hormone. Continued analysis of the aquaporins is providing detailed molecular insight into the fundamental physiological problems of water balance and water balance disorders.

Animals↗
PubMed1995

Aquaporin-1 water channels in short and long loop descending thin limbs and in descending vasa recta in rat kidney.

The localization of aquaporin-1 water channels (AQP-1) in nephron and vascular structures in rat kidney were characterized, because vascular bundles are known to play a key role in urinary concentration. Immunohistochemistry and immunoelectron microscopy were applied on thin cryosections or ultrathin Lowicryl sections, using an optimized freeze-substitution method. Within the vascular bundles, AQP-1 is localized in descending thin limbs (DTL) of short nephrons in apical and basolateral membranes. The expression in DTL of short nephrons is considerably lower compared with the expression in long nephrons, consistent with the known lower osmotic water permeability of this segment. Furthermore, DTL of short nephrons expressing AQP-1 continue abruptly into a thin limb segment without AQP-1. This suggests the existence of a novel thin limb epithelium in the outer medulla. Extensive expression of AQP-1 is observed in apical and basolateral membranes of DTL of long nephrons, which are localized in the periphery of the vascular bundles. The expression decreases along the axis of long nephron DTLs in correlation with the known water permeability characteristics of thin limb segments. DTLs of both short and long nephrons continue abruptly into thin limb segments without AQP-1 expression, revealing an abrupt cell-to-cell transition. In vasa recta, AQP-1 is selectively localized in the nonfenestrated endothelium of descending vasa recta, whereas the fenestrated endothelium of ascending vesa recta and peritubular capillaries do not express AQP-1. AQP-1 is localized in both apical and basolateral plasma membranes, which is logical for transendothelial water transport. Isolated perfused descending vasa recta display high water permeability, and, unlike sodium permeability, diffusional water permeability is partly inhibited by mercurials, thus substantiating the presence of mercurial-sensitive water channels in descending vasa recta. Thus AQP-1 is localized in DTL and descending vasa recta within vascular bundles, and AQP-1 expression in DTL segments is in exact concordance with the known water permeability characteristics, strongly supporting that AQP-1 is the major constitutive water channel of the nephron.

Animals↗