Oretta Murri

Different modulation of aromatase activity in frog testis in vitro by ACE and ANG II

The aim of the present research was to study the role of angiotensin-converting enzyme (ACE) and ANG II in amphibian ( Rana esculenta) testicular steroidogenesis and prostaglandin production. Hormonal effects of ACE, ACE inhibitors, synthetic bullfrog ANG I, and [Val5]ANG II were determined in frog testis of prereproductive period. Production of 17β-estradiol, progesterone, androgens, and PGE2 and PGF2α was determined by incubating frog testes with ACE (2.5 mU/ml), captopril (0.1 mM), lisinopril (0.1 mM), [Val5]ANG II (1 μM), and synthetic bullfrog ANG I (1 μM). The analysis of the data showed an independent modulation of 17β-estradiol and androgen production by ACE and ANG II. The ACE pathway caused a decrease of 17β-estradiol production and an increase of androgen production in frog testes; on the other hand, the ANG II pathway increased 17β-estradiol production and decreased androgen production. The determination of testicular aromatase activity showed a positive regulation by ANG II and a negative regulation by ACE. As for prostaglandin production, only ANG II influenced PGF2α. These results suggest a new physiological role of ACE and ANG II in modulating steroidogenesis and prostaglandin production.The aim of the present research was to study the role of angiotensin-converting enzyme (ACE) and ANG II in amphibian (Rana esculenta) testicular steroidogenesis and prostaglandin production. Hormonal effects of ACE, ACE inhibitors, synthetic bullfrog ANG I, and [Val(5)]ANG II were determined in frog testis of prereproductive period. Production of 17beta-estradiol, progesterone, androgens, and PGE(2) and PGF(2alpha) was determined by incubating frog testes with ACE (2.5 mU/ml), captopril (0.1 mM), lisinopril (0.1 mM), [Val(5)]ANG II (1 microM), and synthetic bullfrog ANG I (1 microM). The analysis of the data showed an independent modulation of 17beta-estradiol and androgen production by ACE and ANG II. The ACE pathway caused a decrease of 17beta-estradiol production and an increase of androgen production in frog testes; on the other hand, the ANG II pathway increased 17beta-estradiol production and decreased androgen production. The determination of testicular aromatase activity showed a positive regulation by ANG II and a negative regulation by ACE. As for prostaglandin production, only ANG II influenced PGF(2alpha). These results suggest a new physiological role of ACE and ANG II in modulating steroidogenesis and prostaglandin production.

Different modulation of steroidogenesis and prostaglandin production in frog ovary in vitro by ACE and ANG II

Our aim was to study the role of angiotensin-converting enzyme (ACE) and angiotensin II (ANG II) on ovarian steroidogenesis and prostaglandin production of amphibian. Hormonal effects of ACE, ACE inhibitors, synthetic bullfrog angiotensin I (ANG I), and [Val5]ANG II were compared on frog ovaries of postreproductive and prereproductive periods. Very high ACE activity was found in ovary of water frog ( Rana esculenta) compared with other frog tissues, and this activity was inhibited by the typical ACE inhibitors, captopril and lisinopril. Frog ovary tissue in postreproductive and prereproductive periods was incubated in vitro in the presence of ACE (2.5 mU/ml), captopril (0.1 mM), lisinopril (0.1 mM), [Val5]ANG II (1 μM), and synthetic bullfrog ANG I (1 μM). Production of 17β-estradiol, progesterone, androgens, and prostaglandins E2 and F2α was determined. The data showed a modulation of 17β-estradiol, progesterone, and prostaglandin E2 production by ovary ACE; on the other hand, [Val5]ANG II modulated the production of progesterone and prostaglandin F2α, whereas androgen production was not influenced. The present in vitro studies suggest the existence of two pathways independently regulated by ACE and ANG II modulating ovarian steroidogenesis and prostaglandin production.

Degradation of thymic humoral factor γ2 by human plasma: involvement of angiotensin converting enzyme

The degradation of thymic humoral factor-gamma2 (THF-gamma2), an immunoregulatory octapeptide important for T-lymphocyte regulation, by enzymes present in human plasma, was investigated. THF-gamma2 was metabolized through two steps that involved the detaching of N-terminal amino acid leucine followed by hydrolysis of the Lys(6)-Phe(7) bond. The THF-gamma2 cleavages were sensitive to aminopeptidase and metalloproteinase inhibitors. The degradation was completely blocked by amastatin and specific inhibitors of angiotensin converting enzyme (ACE). The cleavages occurred independently, with two different kinetics, faster for the N-terminal hydrolysis than for that of the Lys(6)-Phe(7) bond. Purified human plasma ACE was used to characterize the hydrolysis of Lys(6)-Phe(7) bond. The K(m) and K(cat) values for THF-gamma2 hydrolysis were 0.273 mM and 107 s(-1), respectively. The optimum of chloride concentration was 300 mM, while that of pH was 7.6. The presence of ACE in circulating mononuclear cells raises the possibility that it may play a role in modulating the THF-gamma2 activity.

Purified angiotensin converting enzyme from Rana esculenta ovary influences ovarian steroidogenesis in vitro.

The aim of the present study was to purify and characterize angiotensin-converting enzyme (ACE) present in frog ovary (Rana esculenta). Detergent and trypsin-extracted enzymes were purified using a one-step process, consisting of affinity chromatography on lisinopril coupled to Sepharose 6B. The molecular mass was 150 kDa for both detergent-extracted and trypsin-extracted enzyme. The specific activity of detergent-extracted and trypsin-extracted ACE was 294 U mg−1 and 326 U mg−1 respectively. The optimum pH range was from 7–8.5 at 37°C and the optimum temperature was 50 °C. Optimum chloride concentration was about 200 mM for synthetic substrate FAPGG (N-[3-(2-furyl)acryloyl] L-phenylalanyl glycyl glycine) and angiotensin I, and 10 mM for bradykinin. The Km and Kcat values for FAPGG were 0.608±0.07 mM and 249 sec−1 respectively and I50 values for captopril and lisinopril, two specific ACE inhibitors, were 68±12.55 nM and 6.763±0.66 nM respectively. Frog ovary tissue from prereproductive period was incubated in vitro in the presence of frog ovary ACE (2.5 mU/ml), captopril (0.1 mM), and lisinopril (0.1 mM). Production of 17β-estradiol, progesterone, and prostaglandins E2 and F2α was determined. The data showed a modulation of 17β-estradiol, progesterone and prostaglandin E2 production by ovary ACE.ResumenLa finalidad de este estudio es la purificación y caracterización de la enzima convertidora de angiotensina (ACE) contenida en el ovario de rana (Rana esculenta). La enzima, extraída tanto con detergente como con tripsina, se purificó mediante un proceso único, consistente en una cromatografía de afinidad con lisinopril ligado a Sepharose 6B. El peso molecular de la enzima resultó ser de 150 kDa, tanto por la extracción con detergente como con tripsina. La actividad específica de la ACE extraída con detergente y tripsina fue de 294 U mg−1 y 326 U mg−1, respectivamente. El intervalo de pH óptimo fue de 7–8,5 a 37°C y la temperatura óptima, de 50 °C. La concentración molar óptima de cloruro fue sobre 200 mM para el sustrato sintético FAPGG (N-[3-(2-furyl) acryloyl] L-phenylalanyl glycyl glycine) y angiotensina I, y 10 mM para la bradiquinina. Los valores de Km y Kcat para FAPGG fueron 0,608±0,07 mM y 249 sec−1, respectivamente, y los valores de I50 para captopril y lisinopril, dos inhibidores específicos de ACE, fueron 68±12,55 nM y 6,763±0,66 nM, respectivamente. Tejido ovárico de rana en períodos prerre-productivos se incubó in vitro en presencia de ACE de ovario de rana (2,5 mU/ml), captopril (0,1 mM), y lisinopril (0,1 mM), determinán-dose la producción de 17β-estradiol, progesterona y de prostaglandinas E2 y F2α. Los resultados muestran modulación de la producción de 17β-estradiol, progesterona y prostaglandinas E2 por ACE ovárica.

Synthetic seminal plasma peptide inhibits testosterone production in frog testis in vitro

The role of synthetic seminal plasma peptide, designed using biochemical and mass spectroscopy analyses of native peptides extracted from seminal plasma, was studied in amphibian (Rana esculenta) testicular steroidogenesis. Production of testosterone and prostaglandin F(2alpha) was determined by incubating frog testes with synthetic peptide in vitro. Analysis of the data showed a dose-dependent inhibition of testosterone production (43% at 10(-5) M concentration) without prostaglandin F(2alpha) synthesis being affected. Determination of the peptide activity during the annual R. esculenta reproductive cycle showed inhibition of testosterone production in post-reproductive and recovery periods, suggesting a possible involvement of peptide in gonad steroidogenesis.

Synthesis and characterization of tetramethylrhodaminethiocarbamoyl-(Glu1)–epidermal mitosis-inhibiting pentapeptide

A fluorescent analog of epidermal mitosis-inhibiting pentapeptide (pGlu-Glu-Asp-Ser-Gly) was synthesized by reacting tetramethylrhodamine isothiocyanate with ring-opened epidermal mitosis-inhibiting pentapeptide. The ring-opening reaction of the pyrrolidone moiety was performed with mild acidic hydrolysis and the product purified by reversed-phase high-performance liquid chromatography. Tetramethylrhodaminethiocarbamoyl-(Glu(1))-epidermal mitosis-inhibiting pentapeptide was purified by chromatography on Sephadex G-25 and reversed-phase high-performance liquid chromatography. After characterization by amino acid analysis, the analog was incubated in presence of A431 cell line to visualize the cellular localization of the epidermal mitosis-inhibiting pentapeptide. The data gave negative results.