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Background: Asymmetric dimethylarginine (ADMA) inhibits the production of nitric oxide, a key regulator of the vascular tone, and may be important in the development of cardiovascular disease (CVD). Our aim was to reliably quantify the association of ADMA and its isomer symmetric dimethylarginine (SDMA) with the risk of CVD outcomes in long-term cohort studies., Methods and Results: Data were collated from 22 prospective studies involving a total of 19 842 participants, which have recorded 2339 CVD, 997 coronary heart disease, and 467 stroke outcomes during a mean follow-up of 7.1 years. In a comparison of individuals in the top with those in the bottom third of baseline ADMA values, the combined risk ratios were 1.42 (95% confidence interval: 1.29 to 1.56) for CVD, 1.39 for coronary heart disease (1.19 to 1.62), and 1.60 for stroke (1.33 to 1.91). Broadly similar results were observed according to participants' baseline disease status (risk ratios for CVD: 1.35 [1.18 to 1.54] in general populations; 1.47 [1.16 to 1.87] in individuals with pre-existing CVD; and 1.52 [1.26 to 1.84] in individuals with pre-existing kidney disease) and by different study characteristics, including geographical location, sample type, assay method, number of incident outcomes, and level of statistical adjustment (all P values>0.05). In contrast, in 8 prospective studies involving 9070 participants and 848 outcomes, the corresponding estimate for SDMA concentration was 1.32 (0.92 to 1.90) for CVD., Conclusions: Available prospective studies suggest associations between circulating ADMA concentration and CVD outcomes under a broad range of circumstances. Further research is needed to better clarify these associations, particularly in large general population studies., (© 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

The urinary bladder of the aquatic toad Xenopus laevis is known to exhibit a low permeability to water and a poor sensitivity to antidiuretic hormone. In order to precise the characteristics and the specific cellular mechanisms of this reduced hydro-osmotic response we used a sensitive volumetric technique to monitor net water flow and studied the correlation between the antidiuretic hormone (ADH)-induced net water flow and the fine ultrastructural appearance of the urinary bladder epithelium. Transmural net water flow was entirely dependent on the osmotic gradient across the preparation and not on the hydrostatic pressure difference. We observed the existence of a low but significant hydro-osmotic response to arginine vasopressin. Freeze-fracture electron microscopy demonstrated the presence of typical aggrephores in the subapical cytoplasm. The response to the hormone was accompanied by the appearance of typical intramembrane aggregates into the apical plasma membrane. Water permeability increase and apical aggregate insertion were both slowly but fully reversible. Except for the multilayered structure of the epithelium and the particularly low response to antidiuretic hormone, all the studied permeability and ultrastructural characteristics of the bladder were thus very similar to those observed in other sensitive epithelia such as the amphibian bladder and skin and the mammalian collecting duct which exhibit a high hydro-osmotic response to the hormone.

Several experimental conditions such as antidiuretic hormone (ADH) challenge, apical treatment with phorbol myristate acetate (PMA), and mechanical stretching of the tissue are known to increase the insertion of intramembrane particle aggregates and/or granule exocytosis at the apical border of epithelial cells of amphibian urinary bladders. A constant release of 2 peptides of 76 and 14 kDa apparent molecular mass, respectively, was associated with these treatments. The localization of these 2 polypeptides was assessed by immunofluorescence and electron microscopy immunocytochemistry using fluorescent, peroxidase, and colloidal gold probes. The 76 kDa polypeptide appeared to be associated with the cell coat and with the granule content which is released at the apical cell surface. The 14 kDa peptide was also found in the cell coat, and postembedding immunocytochemistry indicates its presence in cytoplasmic subapical vesicles (aggrephores and/or granules). The migration of these 76 and 14 kDa polypeptides in SDS-polyacrylamide gel electrophoresis was modified neither by a treatment at 90 degrees C, nor by the presence or absence of calcium in the medium. Treatment with EGTA did not modify the fluorescence emission of the two peptides and, consequently, they are probably not among the major calcium binding proteins. The addition to the mucosal medium of the stretch extract or of antibodies raised against the 76 and 14 kDa peptides did not modify ADH-induced water permeability. However, a significant decrease of the hydrosmotic response to ADH occurred in subsequent stimulation-washout cycles when the anti-14 kDa peptide antiserum was applied to the mucosal bath. When the bladders were incubated with a stretch extract, we observed a slight alteration of the short-circuit current (Isc), an increase of the basal Na+ transport, and a decrease of the maximal Isc in response to ADH. The 76 kDa protein, released in the apical medium, could play a protective role in the cellular plasma membrane and could participate in the formation of the thick cell coat lining the apical membrane of the granular cells. The 14 kDa protein might be one of the proteins associated with the aggregates, but further studies will be necessary to clarify its exact role in the ADH-induced permeability modifications observed in amphibian urinary bladders.

It is now generally accepted that ADH-induced increase in water permeability in responsive epithelia is associated with the insertion of specific structures in the apical membrane of epithelial cells. Up to now, these structures have only been recognized in freeze-fractured preparations and their chemical nature is still unknown. In this study, we used the label-fracture method (Pinto da Silva and Kan, J. Cell Biol., 99, 1156-1161, 1984) to investigate the distribution of wheat germ agglutinin (WGA) on the luminal plasma membrane of freeze-fractured frog urinary bladder epithelial cells. With label-fracture, the cytochemical markers are seen superimposed with the conventional high resolution image of the E face. Label-fracture of tissue treated for 15 min with WGA and subsequently labeled with colloidal gold coated with ovomucoid showed uniform distribution of gold particles along the exoplasmic fracture face. Stereomicrographs show that the gold label is under the fracture face as it is attached to the outer surface of the membrane. Preincubation of the bladder with WGA for 3 hr induced a segregation of the intramembranous particles of the apical plasma membrane. In this condition, we observed a co-distribution of WGA-gold complexes with the segregated particles on the E face. This indicates that WGA-binding sites are located on glycoproteins which probably comprise the large intramembranous particles dispersed on the exoplasmic faces of freeze-fractured luminal membranes. In contrast, the numerous small intramembrane particles observed on P faces remained evenly distributed even after exposure to WGA and are, therefore, unrelated to WGA receptor sites. After WGA treatment, ADH still induced the formation of aggregates inside the smooth domains. A few WGA-binding sites appeared to be associated to these aggregates.

The apical plasma membrane of epithelial cells of frog and toad urinary bladder is subject to large modifications during the induction of water permeability by the antidiuretic hormone. A better characterization of the apical membrane is necessary for a clear understanding of the mechanisms of hormone action. Towards this end, apical material was extracted by enzymatic treatment and by incubation with detergent. Proteolytic enzyme alone had little effect under our conditions. A pretreatment with several glycosidases (alpha-mannosidase or endo-beta-N-acetylglucosaminidase H) increased the hydrolytic action of papain, elastase, proteinase K or Staphylococcus aureus V8 protease and allowed the detection of a major 76 kD in SDS gel electrophoresis. The n-octyl-beta-D-glucopyranoside (0.2%) led to the extraction after 150 mn of 1 to 5 micrograms proteins per cm2 of amphibian urinary bladder apical surface. The extracted proteins migrated as several bands on SDS gels. One of them probably corresponds to the 76 kD fragment obtained after proteolysis. The absence of alteration of the water permeability after extraction and the good preservation of the ultrastructure are evidence for the localisation of the 76 kD at the apical membrane surface. This protein may be the best candidate as antigen to raise antibodies against the apical surface of amphibian urinary bladder epithelial cells.