Angiotensin-converting enzyme inhibition reduces lipid deposits in myocardium and improves left ventricular function of obese zucker rats

Objective: Alterations in the renin angiotensin system, cardiac lipotoxicity, and left ventricular (LV) dysfunction have been reported in obese rats. The present study examined whether angiotensin‐converting enzyme inhibition could ameliorate lipid deposition and ventricular function in the myocardium of obese Zucker rats (OZRs). Research Methods and Procedures: For 6 months, rats were treated as follows: Group (G) 1, OZR, no treatment; G2, OZR + ramipril (R); G3, OZR + amlodipine (AML); and G4, lean Zucker rats. LV function was assessed by echocardiogram and lipid deposits in cardiomyocytes (LDCM) by light microscopy using Oil red O. Results: At the end of the experiment, both OZR + R and OZR + AML groups presented similar reduction in blood pressure in comparison with untreated OZR (p < 0.01). OZR with R presented lower insulin‐to‐glucose ratio and lower serum triglycerides and cholesterol when compared with both untreated OZR and OZR with AML (p < 0.01). Fractional shortening by echocardiogram was as follows: G1, 25.4 ± 3.8 (vs. G2 and G4, p < 0.05); G2, 37.2 ± 2.4; G3, 29.3 ± 4.4 (vs. G2 and G4, p < 0.05); and G4, 40.8 ± 2.3. Percentage LDCM was as follows: G1, 12.4 ± 2.7 (vs. G2 and G4, p < 0.05); G2, 0.8 ± 0.2; G3, 11.1 ± 2.1 (vs. G2 and G4, p < 0.05); and G4, 0.1 ± 0.1. There was a negative correlation between fractional shortening and LDCM percentage in OZR (r = ?0.93) and in OZR + AML (r = ?0.87). Discussion: AML reduced blood pressure significantly; however, it failed to modify both metabolic parameters and LDCM. In contrast, R showed a substantial reduction in LDCM, together with LV function preservation.     

Error-prone EF-Tu reduces in vivo enzyme activity and cellular growth rate

Mutations in Salmonella typhimurium encoding error-prone EF-Tu (tufA8, tufB103) enhance translational error levels and also cause a reduced growth rate. The relative changes in error level and growth rate are inversely related and dependent on the status of the two tuf genes. Possible causes of the reduced growth rate were investigated. Several important parameters with the potential to alter growth rate (the EF-Tu-ribosome interaction, the in vivo elongation rate and the processivity of translation), are all relatively unaffected by the tuf mutations. The small reduction in processivity observed in some strains is not quantitatively related to the growth rate reduction. Instead, the error-enhancing mutations are associated with a large reduction in the specific activity of a test protein, beta-galactosidase, suggesting by inference that the reduced growth rate is a consequence of the synthesis of error-containing proteins.     

Angiotensin-converting enzyme inhibition attenuates myonuclear addition in overloaded slow-twitch skeletal muscle

Because optimal overload-induced skeletal muscle hypertrophy requires ANG II, we aimed to determine the effects of blocking ANG II production [via angiotensin-converting enzyme (ACE) inhibition] on potential mediators of hypertrophy in overloaded skeletal muscle, namely, myonuclear addition and fibroblast content. In a 2 x 2 design, adult (200-225 g) female Sprague-Dawley rats were placed into one of four groups (n = 8/group): 7-day skeletal muscle overload, sham operation, 7-day skeletal muscle overload with ACE inhibition, or sham operation with ACE inhibition. Functional overloads of the plantaris and soleus muscles were produced via bilateral surgical ablation of the synergistic gastrocnemius muscle, and ACE inhibition was accomplished by the addition of the ACE inhibitor enalapril maleate to the animals' daily drinking water (0.3 mg/ml). Myonuclear addition and extrasarcolemmal nuclear proliferation, as measured by in vivo 5-bromo-2'-deoxyuridine labeling, were significantly (P < or = 0.05) increased by overload in both the slow-twitch soleus and fast-twitch plantaris muscles. Furthermore, ACE inhibition attenuated these overload-induced increases in the soleus muscle but not in the plantaris muscle. However, the effect of ACE inhibition on soleus extrasarcolemmal nuclei was not likely due to differences in fibroblast content because overload elicited significant increases in vimentin-positive areas in soleus and plantaris muscles, and these areas were unaffected by ACE inhibition in either muscle. There was no effect of ACE inhibition on any measure in sham-operated muscles. Collectively, these data indicate that ANG II may mediate the satellite cell response to overload in slow-twitch soleus but not in fast-twitch plantaris muscles and that this effect may occur independently of changes in fibroblast content.     

Calpain inhibition decreases the growth rate of mammalian cell colonies

The calpains, a family of calcium-requiring intracellular proteases, are proposed regulators of cell proliferation. However, ablation of the calpain small subunit gene necessary for function of the conventional calpains did not result in decreased rate of proliferative growth of mouse stem cells under routine culture conditions. To address the reasons for this discrepancy, Chinese hamster ovary cell lines were established that overexpress the calpain inhibitor protein, calpastatin, under control of the ecdysone congener, ponasterone A. Overexpression of calpastatin in these cell lines resulted in a decreased growth of isolated colonies adhering to tissue culture plates. However, when cells were plated at higher density, calpastatin overexpression had no influence on proliferative growth rate. Growth of colonies in soft agar was not inhibited by calpastatin overexpression. Cell adhesion, cell de-adhesion, and cell motility all appeared to be normal after calpastatin overexpression. Differential display analysis was initiated to detect possible alteration of gene expression upon calpastatin overexpression. Analysis of approximately 3000 differential display PCR signals resulted in identification of one band that was underexpressed. Northern blot analysis confirmed a decreased amount of approximately 1 kb mRNA in cells overexpressing calpastatin. Sequence analysis identified a putative protein, Csr, containing a region homologous to two ubiquitin transferases and a putative cation channel protein.     

Probing the basis of domain-dependent inhibition using novel ketone inhibitors of Angiotensin-converting enzyme

Human angiotensin-converting enzyme (ACE) has two homologous domains, the N and C domains, with differing substrate preferences. X-ray crystal structures of the C and N domains complexed with various inhibitors have allowed identification of active site residues that might be important for the molecular basis of this selectivity. However, it is unclear to what extent the different residues contribute to substrate domain selectivity. Here, cocrystal structures of human testis ACE, equivalent to the C domain, have been determined with two novel C domain-selective ketomethylene inhibitors, (5 S)-5-[( N-benzoyl)amino]-4-oxo-6-phenylhexanoyl- l-tryptophan (kAW) and (5 S)-5-[( N-benzoyl)amino]-4-oxo-6-phenylhexanoyl- l-phenylalanine (kAF). The ketone groups of both inhibitors bind to the zinc ion as a hydrated geminal diolate, demonstrating the ability of the active site to catalyze the formation of the transition state. Moreover, active site residues involved in inhibitor binding have been mutated to their N domain counterparts, and the effect of the mutations on inhibitor binding has been determined. The C domain selectivity of these inhibitors was found to result from interactions between bulky hydrophobic side chain moieties and C domain-specific residues F391, V518, E376, and V380 (numbering of testis ACE). Mutation of these residues decreased the affinity for the inhibitors 4-20-fold. T282, V379, E403, D453, and S516 did not contribute individually to C domain-selective inhibitor binding. Further domain-selective inhibitor design should focus on increasing both the affinity and selectivity of the side chain moieties.     

Angiotensin-converting enzyme and anorexia nervosa

Angiotensin-converting enzyme (ACE) activity was measured in 10 patients with anorexia nervosa, 6 with hyperthyroid Graves' disease, and 7 with primary hypothyroidism. Patients with anorexia nervosa had a low serum ACE activity (9.8 +/- 2.2 IU/l), as compared to findings in normal subjects (13.4 +/- 3.5 IU/l) (P less than 0.05). Patients with hyperthyroid Graves' disease had high serum ACE activity (23.7 +/- 5.8 IU/l), as compared to levels in normal subjects (P less than 0.01), and patients with primary hypothyroidism tended to have low serum ACE activity (10.1 +/- 1.8 IU/l), compared to the normal subjects (P less than 0.1). Following weight gain (before; 71.3 +/- 10.2% of ideal body weight, after; 88.7 +/- 5.6% of ideal body weight), serum ACE activity in patients with anorexia nervosa reverted to within the normal range (13.8 +/- 3.5 IU/l), and serum T3 concentration was restored to the normal range (before; 0.7 +/- 0.2 ng/ml, after; 1.1 +/- 0.3 ng/ml). In these patients, ACE activity correlated with the per cent of ideal body weight (P less than 0.05). These data suggest that, in underweight subjects with anorexia nervosa, decreased serum ACE activities may relate to emaciation.     

Angiotensin-converting enzyme activity decreases during fasting

Serum angiotensin-converting enzyme (ACE) activity varies directly with thyroid hormone levels in states of altered thyroid function. Because T3 levels decrease during fasting, ACE activity was examined to ascertain if it was reduced in this low T3 condition. Eighteen obese euthyroid subjects were hospitalized and placed on a weight-maintaining diet for 4 days. Nine subjects (Group 1) underwent a fast (50 kcal/day) for 8 days. Nine (Group 2) subjects received T3 (5 micrograms q 3 h) during an identical fast. Weight loss was identical in both groups (-4.4 kg). Serum T3 fell in Group 1 from 104 +/- 8 to 50 +/- 4 ng/d/(p less than .05) but was unchanged in Group 2 (114 +/- 11 ng/dl fed vs. 120 +/- 14 ng/dl fasted). Blood pressures fell significantly in Group 1 (mean systolic: 112----104 mmHg; diastolic: 71----65 mmHg, p less than 0.05), but not in Group 2 subjects. ACE activity fell progressively in Group 1 subjects during fasting (14.4 +/- 1.6 U/ml fed vs. 12.8 +/- 1.4 U/ml fasted p less than 0.05). ACE activity was not decreased significantly early in the fast in patients given T3, but by late fast (days 6-8) was reduced to the same degree as in Group 1 subjects. Glucose and insulin levels fell similarly in both groups. Conclusions: (1) ACE activity is reduced during starvation. This effect is not mediated by T3. (2) Blood pressure reduction during fasting may result from the low T3 levels, but not from decreased ACE activity. Interpretation of serum ACE activity must be viewed in the context of a patient's diet.     

Angiotensin-converting enzyme as a target for the development of novel insect growth regulators

Insect angiotensin converting enzyme (ACE) is a zinc metallopeptidase capable of inactivating a variety of small to medium size peptide hormones by cleavage of C-terminal dipeptides and dipeptideamides. High levels of ACE activity are found in the hemolymph and in reproductive tissues of insects, where the enzyme is considered to have an important role in the metabolism of bioactive peptides. Therefore, inhibiting ACE activity is expected to interfere with the peptidergic endocrine system and to have detrimental effects on growth, development and reproduction. We will review the studies showing that ACE inhibitors do indeed disrupt growth and reproduction in various insect species. We will also present some new genetic and pharmacological data that strengthens our conclusion that ACE should be considered as a potential target for the development of new insect growth regulators.     

Angiotensin-converting enzyme inhibition studies by natural leech inhibitors by capillary electrophoresis and competition assay.

A protocol to follow the processing of angiotensin I into angiotensin II by rabbit angiotensin-converting enzyme (ACE) and its inhibition by a novel natural antagonist, the leech osmoregulator factor (LORF) using capillary zonal electrophoresis is described. The experiment was carried out using the Beckman PACE system and steps were taken to determine (a) the migration profiles of angiotensin and its yielded peptides, (b) the minimal amount of angiotensin II detected, (c) the use of different electrolytes and (d) the concentration of inhibitor. We demonstrated that LORF (IPEPYVWD), a neuropeptide previously found in leech brain, is able to inhibit rabbit ACE with an IC(50) of 19.8 micro m. Interestingly, its cleavage product, IPEP exhibits an IC(50) of 11.5 micro m. A competition assay using p-benzoylglycylglycylglycine and insect ACE established that LORF and IPEP fragments are natural inhibitors for invertebrate ACE. Fifty-four percent of insect ACE activity is inhibited with 50 micro m IPEP and 35% inhibition with LORF (25 mm). Extending the peptide at both N- and C-terminus (GWEIPEPYVWDES) and the cleavage of IPEP in IP abolished the inhibitory activity of both peptides. Immunocytochemical data obtained with antisera raised against LORF and leech ACE showed a colocalization between the enzyme and its inhibitor in the same neurons. These results showed that capillary zonal electrophoresis is a useful technique for following enzymatic processes with small amounts of products and constitutes the first evidence of a natural ACE inhibitor in invertebrates.     

Angiotensin-converting enzyme and cardiovascular disease risk.

The published studies of the association of the angiotensin-converting enzyme (ACE) genotype with cardiovascular disease have used many different diagnostic criteria for cardiovascular disease and have drawn their samples from different patient groups and different populations. This review examines the association of the ACE DD genotype with cardiovascular disease risk in studies grouped by their case criterion, the geographical region of the population samples, and by the cardiovascular risk level of the patient sample. In studies where the underlying odds ratios are determined to be homogeneous, the overall odds ratios for myocardial infarction and coronary artery disease with regard to the ACE DD genotype are estimated using the Mantel-Haenszel method.     

Inhibition by neuroblastoma growth inhibitory factor of ascites-type neuroblastoma cell growth in coculture with normal glioblasts

A new ascites type neuroblastoma clone (NAs-1), which is characteristic both in anchorage-independent growth and catecholaminergic functions, attached on the monolayer culture of glioblasts and was subjected to morphological differentiation including the extrusion of neuronal processes. Other conventional neuroblastoma cells (Neuro2a, NS-20Y, and N1E-115) as well as NAs-1 in cocultured with normal glioblasts underwent a decrease in cell growth rates and DNA synthesis under the effect of the neuroblastoma growth inhibitory factor (NGIF) produced by glioblasts. After their NGIF production had been reduced by u.v. irradiation, glioblasts lost the growth-inhibitory and differentiation-promoting effects in coculture with NAs-1. The supplement of NGIF into u.v.-treated glioblasts restored the dose-dependent growth inhibition of NAs-1. The addition of nerve growth factor into the coculture system brought about neither the marked effect on growth inhibition of NAs-1 nor the morphological differentiation. The results imply a direct function of NGIF on the paracrine regulation of neuroblastoma cell growth in the coculture with normal glioblasts.