Immunolocalization of angiotensin 1 converting enzyme in the human male genital tract by the avidin-biotin-complex method

Immunoreactivity for Angiotensin 1 Converting Enzyme was investigated in a series of 12 fixed and paraffin-embedded normal human genital tract specimens. The Avidin-Biotin-Complex immunoperoxidase method was used with overnight (12 h) incubation with a polyclonal antihuman kidney Angiotensin 1 Converting Enzyme antiserum. All tissues, including testis, different parts of epididymis, ductus deferens, prostate and seminal vesicles, demonstrated a staining pattern. Immunoreactivity was observed on the luminal surface of these epithelia especially on non-motile stereocilia. An intracellular positivity was only observed in spermatids on the acrosomal cap. Besides, an immunologic identity of Angiotensin 1 Converting Enzyme located on the different epithelia of the human male genital tract, on the endothelial cells of vessels and on the proximal tubule brush border of the kidney was observed.     

Downregulation of angiotensin converting enzyme by TNF-alpha in differentiating human macrophages

OBJECTIVE: To investigate regulation of angiotensin converting enzyme (ACE) by tumour necrosis factor alpha (TNF-alpha) in differentiating human peripheral blood monocytes (PBM). METHODS: Human PBM were allowed to differentiate to macrophages for 0-7 days and ACE amount was measured during differentiation. Experiments with TNF-alpha were performed after 2 days of differentiation. Cell cultures were incubated with TNF-alpha (0.5-10ng/ml) without or with SB 202190 (5microM), or PD 98059 (40microM). ACE amounts were measured by an inhibitor binding assay (IBA) and ACE mRNA levels by RNase protection assay (RPA). Activated p44/42 and p38 MAP kinases were measured by Western Blot analysis using phospho-p44/42 and -p38 MAPK antibodies. RESULTS: ACE amount increased by 40-fold along with macrophage differentiation. TNF-alpha caused dose dependent suppression of the amount of ACE and decreased levels of ACE mRNA. TNF-alpha activated p44/42 and p38 MAP kinases, which was inhibited by the specific inhibitors of these kinases, PD98059 or SB202190, respectively. Pretreatment of the cells with SB 202190, or PD 98059 both partly reversed TNF-alpha induced ACE suppression. CONCLUSIONS: TNF-alpha downregulated ACE, which effect was probably mediated by both p44/42 and p38 MAPK pathways. Local downregulation of ACE by TNF-alpha may be a counterbalancing mechanism in inflammatory processes.     

Neuropeptide Y in the human male genital tract

Neuropeptide Y (NPY) was found in high concentrations in the male genital tract. NPY levels were highest in the seminal vesicles, prostate, corpus cavernosum and vas deferens, where large numbers of immunoreactive nerve fibres were detected. Considerable quantities were also found in the epididymis and spongiosum. Lower concentrations were found in the glans penis, testis and foreskin. The presence of a large number of nerves containing NPY suggest that this active neuropeptide may play a role in control of genital function.     

Epitope mapping of the domains of human angiotensin converting enzyme

Somatic angiotensin converting enzyme (sACE), contains in its single chain two homologous domains (called N- and C-domains), each bearing a functional zinc-dependent active site. The present study aims to define the differences between two sACE domains and to localize experimentally revealed antigenic determinants (B-epitopes) in the recently determined three-dimensional structure of testicular tACE. The predicted linear antigenic determinants of human sACE were determined by peptide scanning ("PEPSCAN") approach. Essential difference was demonstrated between locations of the epitopes in the N- and C-domains. Comparison of arrangement of epitopes in the human domains with the corresponding sequences of some mammalian sACEs enabled to classify the revealed antigenic determinants as variable or conserved areas. The location of antigenic determinants with respect to various structural elements and to functionally important sites of the human sACE C-domain was estimated. The majority of antigenic sites of the C-domain were located at the irregular elements and at the boundaries of secondary structure elements. The data show structural differences between the sACE domains. The experimentally revealed antigenic determinants were in agreement with the recently determined crystal tACE structure. New potential applications are open to successfully produce mono-specific and group-specific antipeptide antibodies.     

Immunolocalization of glycodelin in the genital tract of rats

Glycodelin, also known as placental protein 14 has been predominantly localized to organs of the human genital tract. Unfortunately the physiological role of glycodelin is largely unknown since it depends on limited availability of tissues. Therefore, a suitable animal model to study the role of glycodelin would be desirable. Previously, it was shown that glycodelin mRNA is expressed in the genital tract of male and female rats. In the present study, we demonstrate the expression of glycodelin protein in male and female rats by immunohistochemistry and Western blot analysis. For this purpose a polyclonal antibody was generated against glycodelin peptide. In female rats, glycodelin was found in the epithelial gland cells of the uterus, epithelial cells of the fallopian tube as well as in corpora lutea, interstitial and theca cells of the ovary. Glycodelin was distributed in all epithelial cells of the epididymis and the seminal vesicle. In the seminiferous epithelium, glycodelin was seen in all developmental stages of spermatogonia and spermatocytes and in Sertoli cells. Whereas in the rat male reproductive tract glycodelin expression is slightly different from human or primate tissues, in organs of the rat female genital tract glycodelin expression is similar to humans and primates.     

Inactivation of angiotensin converting enzyme by sodium nitroprusside

Angiotensin I converting enzyme is rapidly inactivated by sodium nitroprusside and that inactivation is suppressed in the presence of chloride ion and by the presence of L-alanyl-L-proline or glycyl-L-tryptophan, which are both competitive inhibitors of its catalytic activity. The inactivation by sodium nitroprusside appears to result from the modification of an unusually reactive lysine residue in or near the active site.     

Activation of angiotensin converting enzyme by monovalent anions

The angiotensin converting enzyme catalyzed hydrolysis of furanacryloyl-Phe-Gly-Gly is activated by monovalent anions in the order C1- greater than Br- greater than F- greater than NO3- greater than CH3COO-. In the alkaline pH region, increasing anion concentrations decrease the KM but do not change the kcat. This behavior is characteristic of an ordered bireactant mechanism in which the anion binds to the enzyme prior to the substrate. At acidic pH values, however, the anion activation is a result of both a decrease in KM and an increase in kcat, implying a bireactant mechanism in which anion and substrate bind randomly. For both the ordered and the bireactant mechanisms the anion serves as an essential activator. The effect of chloride on enzyme activity was studied over the pH range 5-10 under kcat/KM conditions and demonstrates that the apparent chloride binding constant increases from 3.3 mM at pH 6.0 to 190 mM at pH 9.0. The kcat vs. pH profile exhibits two pK values of 5.6 and 9.6, while the variation of KM with pH is characterized by a pK of 8.9 and a 2-fold increase between pH 6.5 and 7.5. The chloride activation of the hydrolysis of furanacryloyl-Phe-Gly-Gly is compared with that of the physiological substrates angiotensin I and bradykinin.     

Induction of angiotensin converting enzyme in human monocytes in culture.

Angiotensin converting enzyme (E.C.3.4.15.1, peptidyl dipeptidase) in circulating human monocytes rose from undetectable or minimal levels $\text{in}\text{vivo}$ to as high as 35.5 nmol/min·mgprotein (>300-fold increase) after 6 or 7 days in culture. Enzyme induction was enhanced by autologous serum and exposure for two days to 0.45 μM dexamethasone. Potent inhibition of enzyme induction by 370 μg/ml of actinomycin D and 1 μM cycloheximide suggested that new messenger RNA and enzyme biosynthesis are involved in the induction. Human monocyte and lung enzyme were similar with respect to EDTA inhibition, CoCl₂ activation and inhibition by an antienzyme antiserum. Human lymphocytes had minimal or undetectable enzyme which was not induced after 4 days in culture.     

Isolation and characterization of angiotensin converting enzyme from human kidney

Angiotensin converting enzyme [EC 3.4.15.1] was solubilized from the membrane fraction of human kidney cortex using trypsin and purified to homogeneity by DEAE-cellulose, hydroxylapatite and DEAE-Sephadex A-50 column chromatographies, preparative isoelectric focusing, and Sephadex G-200 gel filtration. The final recovery of the enzyme was 13.9%. The molecular weight of the enzyme was estimated to be 199,000 by a sedimentation equilibrium method. A value of 170,000 was obtained for the reduced and denatured enzyme by dodecylsulfate-polyacrylamide gel electrophoresis. The enzyme was a glycoprotein consisting of a single polypeptide chain with an isoelectric point of 5.10. Neutral sugar accounted for 13% per weight of the enzyme. The purified enzyme had a specific activity of 96.9 mumol/min/mg protein for hippurylhistidylleucine. The Km value, Kcat value and hydrolytic coefficient (Kcat/Km) of the enzyme for hippurylhistidylleucine were 2.0 mM, 545 s-1 and 273 mM-1 . s-1, respectively. Rabbit antibody against the human kidney converting enzyme inhibited the activities of the enzymes from human lung and serum as equally as that from human kidney, but not those from sheep, dog, or rat sera. The human kidney and lung converting enzymes were immunologically identical on double immunodiffusion analysis.     

Molecular heterogeneity of angiotensin converting enzyme in human cerebrospinal fluid.

Three different molecular forms of angiotensin converting enzyme (ACE) (approximately Mr 150,000, 80,000 and 40,000, respectively), have been recovered from human cerebrospinal fluid. All three enzymes were inhibited by captopril and enalapril and their activity was potentiated by chloride ions. They were capable of degrading Leu-enkephalin-Arg6 and substance -P, but gave no conversion of neurokinin A. In all these aspects, the CSF enzymes were identical with the human pulmonary enzyme. The Mr 40,000 form of ACE is the smallest active form of the enzyme hitherto reported and is likely to represent a fragment of the C-terminal part of native ACE, where its active center is located.     

Purification of bovine angiotensin converting enzyme

A change has been made in the commonly used lisinopril affinity gel procedure for purifying angiotensin converting enzyme. The new method greatly decreases the time required and greatly increases the yield of pure enzyme. All of the enzyme in various bovine tissues was extracted with 0.5% triton X-100 and applied to the affinity column; 70% was trapped and all of the trapped enzyme was released as the apoenzyme by EDTA. The holoenzyme was recovered by dialysis against zinc containing buffer. The turnover numbers were precisely the same for enzyme from lung, atrium, kidney, striatum and blood. The tissue concentrations of ACE were very different but the final specific activities were the same.     

Renin, kallikrein and angiotensin converting enzyme in human physical loading

The authors studied the correlation between the activity of renin, angiotensin-converting enzyme, the activity of prekallikrein and the blood prekallikrein level in men performing veloergometric exercise. At the same time they recorded the hemodynamic parameters (systolic and diastolic arterial pressure, the systolic rate, stroke volume and cardiac index, specific peripheral resistance). The blood samples collected before and immediately after the exercise showed a 41.4%-increase in the activity of renin and a 95%-increase in that of kallikrein, whereas the level of prekallikrein and the activity of the converting enzyme declined by 19 and 13% (P less than 0.05). These changes were accompanied by an increase in the systolic rate (by 107%) and in the systolic arterial pressure (by 36.7%), as well as by a reduction of the specific peripheral resistance (by 41.4%).     

Inhibition of angiotensin converting enzyme by phosphoramidates and polyphosphates

N alpha-Phosphoryl-L-alanyl-L-proline is a reversible competitive inhibitor of angiotensin converting enzyme with a Ki of 1.4 nM. Alkylation of one phosphate oxygen with methyl, ethyl, or benzyl does not change the Ki. The high activity of the O-alkylated inhibitors demonstrates that the two phosphate oxygen anions do not constitute a bidentate ligand of the active site zinc ion. Substitution of valyltryptophan, glycylglycine, or delta-aminovaleric acid for alanylproline in the phosphoramidate raises the Ki to 12 nM, 25 microM, and 178 microM, respectively. Methylation of the alanine nitrogen in phosphorylalanylproline raises the Ki to 29 microM. Polyphosphates inhibit converting enzyme with the following Ki's: phosphate, approximately 300 mM; pyrophosphate, 2 mM; tripolyphosphate, 18 microM; tetrapolyphosphate, 150 microM. The inhibition by tripolyphosphate appears to be competitive and is unaffected by the addition of excess zinc ion. Since the Ki of tripolyphosphate is nearly 10-fold lower than that of N-phosphoryl-delta-aminovaleric acid and is near that of N alpha-phosphorylglycylglycine, its terminal phosphates may bind the zinc site and the cationic site on the enzyme, thus spanning the S1' and S2' sites.     

Lipopolysaccharides decrease angiotensin converting enzyme activity expressed by cultured human endothelial cells.

Angiotensin converting enzyme (ACE) is present on endothelial cells and plays a role in regulating blood pressure in vivo by converting angiotensin I to angiotensin II and metabolizing bradykinin. Since ACE activity is decreased in vivo in sepsis, the ability of lipopolysaccharide (LPS) to suppress endothelial cell ACE activity was tested by culturing human umbilical vein endothelial cells (HUVEC) for 0-72 hr with or without LPS and then measuring ACE activity. ACE activity in intact HUVEC monolayers incubated with LPS (10 micrograms/ml) decreased markedly with time and was inhibited by 33%, 71%, and 76% after 24 hr, 48 hr, and 72 hr, respectively, when compared with control, untreated cells. The inhibitory effect of LPS was partially reversible upon removal of the LPS and further incubation in the absence of LPS. The LPS-induced decrease in ACE activity was dependent on the concentrations of LPS (IC50 = 15 ng/ml at 24 hr) and was detectable at LPS concentrations as low as 1 ng/ml. That LPS decreased the Vmax of ACE in the absence of cytotoxicity and without a change in Km suggests that LPS decreased the amount of ACE present on the HUVEC cell membrane. While some LPS serotypes (Escherichia coli 0111:B4 and 055:B5, S. minnesota) were more potent inhibitors of ACE activity than others (E. coli 026:B6 and S. marcescens), all LPS serotypes tested were inhibitory. These finding suggest that LPS decreases endothelial ACE activity in septic patients; in turn, this decrease in ACE activity may decrease angiotensin II production and bradykinin catabolism and thus play a role in the pathogenesis of septic shock.     

Metabolism and subcellular localization of angiotensin converting enzyme in cultured human monocytes

The enhancement of monocyte ACE activity during culture by autologous T-lymphocytes was shown to be due to a stimulation of the rate of ACE synthesis. The rate of synthesis increased from 0.020 mU/10(6) monocytes/hr in monocytes cultured alone to 0.063 mU/10(6) monocytes/hr in monocytes co-cultured with T-lymphocytes. The presence of T-lymphocytes during culture did not alter the rate of ACE degradation observed in monocytes cultured alone. The ACE induced in monocytes by T-lymphocytes appears to be an ecto-enzyme. Brief exposure to diazosulfanilic acid (10(-3) M) and papain (250 micrograms/ml) reduced ACE activity 89% and 66%, respectively, without appreciably altering the activity of the cytosolic enzyme, lactate dehydrogenase.     

Spontaneous change of human plasma angiotensin I converting enzyme isoelectric point

The isoelectric point of angiotensin I converting enzyme (ACE) spontaneously changes from 4.3 to 4.6 during purification from human plasma. The spontaneous change in pI corresponds to that occurring with neuraminidase-treated but not with EDTA-treated samples. There is no detectable difference in the molecular weight of, or lectin binding by, the two forms of ACE with different pI's. These data indicate that ACE in the circulation contains a greater amount of sialic acid than purified ACE. The implication is that purified ACE isoenzymes which differ in sialic acid content may not reflect tissue-specific isoenzymes but rather artifacts of purification.     

Regulation of angiotensin converting enzyme activity in cultured human vascular endothelial cells

Angiotensin converting enzyme (ACE) of vascular endothelial cells is suggested to control vascular wall tonus through the conversion of angiotensin I (AI) to angiotensin II (AII) and the degradation of bradykinin. To obtain more insight into the pathophysiological significance of ACE of vascular endothelial cells, we studied the regulation of ACE produced by cultured human umbilical vein endothelial cells (EC). Phorbol 12-myristate 13-acetate (PMA) increased the cellular and medium ACE activity, accompanied by a marked morphological change in EC. N'-O'-dibutylyladenosine 3';5'-cyclic monophosphate (db-cAMP) increased only the cellular ACE activity and not the medium ACE activity. The effect of isoproterenol with 0.1mM theophylline mimicked that of db-cAMP. These findings suggest that PMA and cAMP-related agents participate in the control of vascular wall tonus through the positive regulation of ACE produced by vascular endothelial cells.