Nitric oxide was measured by methemoglobin method

of ACE beyond blood pressure regulation, especially in kidney functions, development and male fertility. In addition to these functions, ACE plays roles in fat metabolism, inflammation and immune responses. Our previous studies using transgenic ACE expression in Ace-/- mice demonstrated tissue- and isoformspecific physiological functions of ACE. The Ts mice, which express transgenic sACE only in the vascular endothelial cells of Ace-/- mice, can restore normal blood pressure. However, gACE is not able to substitute for sACE for the maintenance of normal blood pressure of Ace-/- mice. Expression of gACE, but not sACE, in sperm successfully restores the male fertility of Ace-/mice. ACE is expressed on the cell surface as type I ectoprotein, with a long ectodomain Vitamin E-TPGS biological activity containing the enzymatic active site, short cytoplasmic domain and a transmembrane domain. The ectodomain of ACE can be cleaved by a membrane-bound metalloprotease, to generate a soluble form. Although ACE secretase is unknown, its activity has been characterized as a membrane-associated protease. Previous studies have demonstrated that the cytoplasmic domain of ACE interacts with various signaling proteins such as, Calmodulin, Protein Kinase C, Casein Kinase 2 and regulate the cleavage-secretion of ACE. Moreover, the association of these proteins regulates the phosphorylation of ACE on a specific Ser residue in the cytoplasmic domain. Because both the cell-bound and the secreted forms of ACE are enzymatically active, their functional distinction has been of great interest. Cell-bound Secreted ACE Cannot Restore Normal Blood Pressure ACE, in addition to its enzymatic activity, can act as a signaling protein on the cell surface. Signaling via ACE activates CK2, which phosphorylates the cytoplasmic domain of ACE, suggesting phosphorylation of the cytoplasmic domain of ACE as a potential intracellular event which may contribute to the functions of ACE in the endothelial cells. To investigate the relative roles of secreted and cell-bound ACE in various physiological functions of ACE, we have generated a transgenic mouse model which expressed only the secreted form of sACE, without any cell-bound form. This mutant ACE protein lacks the transmembrane and the cytoplasmic domains and, therefore, is constitutively secreted in the circulation. Our results demonstrated that the secreted ACE could not restore normal blood pressure of Ace-/- mice, although their kidney functions were normal. This study demonstrates that a cell-bound ACE is critical for maintaining normal blood pressure; a secreted ACE cannot substitute for the cell-bound form for this function. approved by the Lerner Research Institute’s Institutional Animal Care and Use Committee. Maintenance of control mice Ace-/- Ts/Ts control mice were generated by mating Ace+/2 Ts/Ts males with Ace-/- Ts/Ts females as described previously. Ace-/- and Ace+/2 control mice were generated by mating Ace+/2 male mice PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19649022 with Ace+/2 female mice. All of the mice described in this study, including Wt, KO, Tie-esACE and Tie-sACE, were of the FVB strain. Southern Blot hybridization Southern blot genotyping was performed as described previously utilizing Sac I digestion of genomic tail snip DNA. Heterozygosis or homozygosis of the transgene was determined by normalizing the transgene value to the endogenous mouse Ace gene value in the same genomic DNA sample using Imagequant software. The endogenous Ace genotype is determined by the presence of a wild-