Effect of ethanol drinking on the gene expression of opioid receptors, enkephalinase, and angiotensin-converting enzyme in two inbred mice strains.

There is convincing evidence that genetic factors contribute to the predisposition to alcoholism. In this respect, alcohol-preferring (like C57BL/6 mice) and alcohol-avoiding lines (like DBA/2 mice) of animals served as models in the search for neurobiological substrates of excessive ethanol consumption. One of the systems that is thought to be associated with the incidence of alcoholism is the endogenous opioid system. In the first experiment, basal mRNA levels of mu- and delta-opioid receptors, and of opioid-degrading enzymes enkephalinase (neutral endopeptidase 24.11; NEP) and angiotensin-converting enzyme (ACE) in the brain regions of C57BL/6 and DBA/2 mice did not reveal genetically determined differences in these parameters between the two strains. Furthermore, in the brain regions studied, the corresponding enzyme activities of NEP and ACE did not differ significantly between the lines of mice, except for a higher NEP activity in the striatum and olfactory bulb of DBA/2 mice (p < 0.01). In the second experiment, C57BL/6 and DBA/2 mice were offered a free choice between water and 10% ethanol solution for 4 weeks and were killed thereafter; from another group, ethanol was removed for 3 days and from a third group ethanol was removed for 3 weeks before killing. In the striatum, a highly significant increase in the ACE mRNA amount was detected after 3 weeks of removal of ethanol in C57BL/6 mice, whereas in DBA/2 mice the delta-opioid receptor mRNA level was increased at this time when compared with the corresponding ethanol treatment group. The most striking changes were seen in the hypothalamus, where mu-opioid receptor, ACE, and NEP mRNA amounts markedly decreased after ethanol treatment in both strains. Thus, chronic ethanol intake caused significant changes in the gene expression of distinct components of the endogenous opioid system. These findings further underline an involvement of the opioid system in the effects of ethanol.

Stimulation of endothelial angiotensin-converting enzyme by morphine via non-opioid receptor mediated processes.

In this study, we examined the influence of morphine and naloxone on the enzymatic activity of different ecto-peptidases located on the surface of endothelial cells. Morphine increased in a concentration dependent manner the degradation of Leu-enkephalin in cultivated bovine aortic endothelial cells. Naloxone, a morphine antagonist, did not prevent this effect, but caused it as well. The enhanced Leu-enkephalin degradation was due to an increase in the activity of angiotensin-converting enzyme (ACE), whereas the activity of other ecto-peptidases (aminopeptidase N and neutral endopeptidase) was not influenced. Despite a high non-specific binding of [3H]-morphine, no specific opioid receptor binding on the endothelial cells could be detected. Autoradiographic investigations with native, cryostat-sectioned cells demonstrated that [3H]-morphine was nearly exclusively located within the nuclei. The present results suggests that the morphine effect concerning ACE activity is not mediated via opioid receptors but presumably by interactions within the cell nucleus.

Gene expression and activity of specific opioid-degrading enzymes in different brain regions of the AA and ANA lines of rats.

There is increasing evidence that alcoholism runs in families suggesting that genetic factors may play a role. In support of this hypothesis, the alcohol-preferring (AA) and the alcohol-avoiding (ANA) rat lines have been developed through selective outbreeding. Numerous studies indicate that the endogenous opioid system may be involved in controlling ethanol consumption. Changes in opioid peptides and opioid receptors have been described after ethanol intake. But, the influence of ethanol on peptidolytic degradation of opioid peptides has been largely ignored, although the peptidase-mediated metabolism of neuropeptides is known as an important regulatory site of peptidergic transmission. Neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE) degrade neuropeptides, including enkephalin and are expressed in the brain. Furthermore, a good correspondence between the regional distribution of NEP and opioid receptors in rat brain has already been reported pointing to a possible role of NEP in regulating opioid peptides. For both enzymes studied, the gene expression pattern was found to be in good agreement with the corresponding enzyme activities in the brain regions investigated, showing the highest levels for both specific mRNAs and enzyme activities in the striatum. Differences in both measured parameters were detected in distinct brain regions of AA and ANA rats. Furthermore, in some brain regions discrepancies between ACE and NEP mRNA levels and the corresponding enzyme activities were observed. For example, in olfactory bulb and striatum such discrepancies were found for both enzymes studied. In tegmentum/colliculi a higher NEP gene expression in AA rats was associated with a higher NEP enzyme activity compared to the amounts found in ANA rats.

Screening of selected basidiomycetes for inhibitory activity on neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE).

Aqueous extracts of 27 basidiomycetes were investigated for their ability to inhibit the activity of angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP). The extracts of 5 fungi inhibited both, ACE and NEP activity, another 18 extracts showed inhibition of the NEP activity whereas only 1 basidiomycete inhibited the ACE activity exclusively. The IC50 values for the ACE inhibition are rather high (between 200 and 1500 micrograms/ml) in comparison to the IC50 of the NEP inhibition (between 40 and 2000 micrograms/ml). These results indicate that the basidiomycetes investigated seem to have a higher potential for the inhibition of the activity of NEP than of ACE. In general, basidiomycetes are a new source for inhibitors of metalloendopeptidases. Resulting from the isolation and characterization of these compounds new leading structures are expectable.

Dexamethasone induced enhanced enkephalin degradation by angiotensin-converting enzyme (ACE) of endothelial cells.

The Leu-enkephalin breakdown by ectopeptidases of cultivated endothelial cells is increased in a concentration-dependent manner by dexamethasone treatment. This glucocorticoid induced selectively the angiotensin-converting enzyme (ACE) of the cells. The activity of the closely related enzyme neutral metalloenopeptidase (NEP) and the aminopeptidase N (APN) also present on the endothelial cell surface were not affected or slightly decreased by dexamethasone. The data indicate the significance of ACE for degradation of Leu-enkephalin during the stress reaction.

The enzymatic degradation of bradykinin in semen of various species.

The degradation of bradykinin in semen and on washed sperm cells of various species (human, pig, cattle, sheep) is mainly controlled by two peptidases, the angiotensin-converting enzyme (ACE/kininase II; E.C. 3.4.15.1) and neutral metalloendopeptidase (NEP; E.C. 3.4.24.11). In addition, minor activities of kininase I (carboxypeptidase N/CPN; E.C. 3.4.17.3) were measured exclusively in human samples. Samples of the investigated species varied considerably in their ratios of the activities of bradykinin degrading peptidases. This should be considered in any approach aimed at maintaining the promoting effect of bradykinin on sperm motility by use of enzyme inhibitors.

Degradation of bradykinin in semen of ram and boar.

The pattern of bradykinin (BK; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9)-inact iva ting peptidases in semen of boar and ram was investigated. The degradation of BK in semen was completely abolished by the metalloprotease inhibitors EDTA and o-phenanthroline. Inhibitors of angiotensin-converting enzyme (ACE; EC 3.4.15.1) and phosphoramidon, an inhibitor of neutral metalloendopeptidase (NEP; EC 3.4.24.11), were only partially effective in preventing BK degradation in semen. An additive effect was seen with simultaneous inhibition of both enzymes, resulting in complete abolition of BK degradation. HPLC analysis demonstrated that exogenous BK in semen is cleaved at Gly4-Phe5, Phe5-Ser6 and Pro7-Phe8. These results indicate that NEP and ACE are the main peptidases responsible for rapid BK inactivation in semen. The involvement of other peptidases known to be responsible for BK cleavage in other tissues and body fluids, namely carboxypeptidase N (EC 3.4.12.7), post proline cleaving enzyme (EC 3.4.21.26) and aminopeptidase P (EC 3.4.11.9) was excluded. NEP and ACE were shown to be localized mainly in seminal plasma and to a lesser extent on sperm cells.

Reduced degradation of biological active peptides during in vitro aging of endothelial cells.

Endothelial cells under in vitro cultivation show an age-dependent decrease in peptide-cleavage activity. After a number of passages, the rates of Leu-enkephalin and bradykinin decomposition by cultivated cells were diminished as compared to their counterparts before in vitro aging. In addition, these cells have been found to be less capable of converting angiotensin I into angiotensin II. Such phenomena could be interpreted as correlated with age-related disturbances concerning the regulation of vascular functions in vitro.

[Tyrosine splitting aminopeptidases on cultivated anterior hypophyseal and vascular endothelial cells]. / Tyrosin-abspaltende Aminopeptidasen an kultivierten Hypophysenvorderlappen- und Gefässendothelzellen.

Activities of aminopeptidases for a tyrosine peptide hydrolysis were characterized with Tyrosyl-7-amino-4-methyl-coumarin as substrate on in vitro cultivated anterior pituitary cells, respectively, on aortic endothelial cells. Furthermore the corresponding activities were measured in different fractions of the cells. The activities of the enzymes in soluble fractions of the cell homogenates are comparable with aminopeptidases of cytosolic compartments of other tissue samples. On the other hand remarkable differences exist between Km- and IC50-values of the membrane preparations of both cell types. Furthermore, the substrate degradation on intact cells by provable membrane bound ectoenzymes is identically for both cell types and this degradation is insensitive for amastatin. Our results are discussed with special respect for the importance of the degradation of biological active peptides with N-terminal tyrosine by aminopeptidases on their physiological targets.

Investigations on the influence of bradykinin upon the motility of ram spermatozoa.

The influence of bradykinin, a component of the kallikrein-kinin system, on the motility of ram spermatozoa was examined in vitro (photometric motility evaluation, penetration test in cervical mucus of sheep, estimation of the percentage of forward-moving spermatozoa in the thermal resistance test). Whereas the motility was found to be influenced by bradykinin the acrosomal status remained undisturbed by it. With fresh as well as with frozen semen, the drug mainly increased the motility of samples with a low initial motility. With frozen semen, the penetration test revealed greater motility rises at 22 degrees C than at 38 degrees C. The importance of the results in relation to the angiotensin-converting enzyme (kininase) as well as the motility evaluation capabilities of the methods used are discussed. There was, however, no positive drug effect after adding the drug in the course of cryopreservation prior to freezing semen in straws although bradykinin solutions frozen in liquid nitrogen maintained their effectiveness.

Angiotensin-converting enzyme and other peptidolytic enzymes in human semen and relations to its spermatologic parameters.

Angiotensin-converting enzyme (ACE) and other enzymes of the renin-angiotensin system (RAS) occur in human semen in high activities. In contrast to bull ejaculates, not all zinc-dependent metallopeptidases are found to be in close correlation to the microscopically determined semen parameters; such a relationship was established only partly for the ACE. On the other hand, the RAS-dependent spermatozoa-bound enzymes, inclusive ACE, uniformly show negative correlations to the spermatologic parameters of human semen. These results, for the first time, point to different functions of the sperm-cell-bound (testicular) and of the seminal plasma (pulmonary) ACE activities.

Occurrence and potential importance of angiotensin-converting enzyme in semen of boars.

The activities of angiotensin-converting enzyme (ACE) and leucinaminopeptidase (LAP) are positively correlated with corresponding concentrations of sperm cells in semen of boars kept under normal conditions. The spermatozoa bound ACE activity, in general, does not reflect differences in the quality of semen (bull and boars). On the other hand, the ACE activity directly bound on the sperm cells is significantly elevated, if 'exogenic noxes' (by feeding or keeping) influence the fertility of boars in a drastic manner. These results are discussed with regard to the differential diagnostic importance for estimating the semen quality and to the causal relations between increased enzyme binding and injury of sperm cells.

Occurrence and potential importance of selected peptidases in bull ejaculates.

The zinc containing peptidases angiotensin-converting enzyme (ACE), neutral metalloendopeptidase (NEP), and leucine aminopeptidase (LAP) occur in bull ejaculates in high activities. These enzyme activities are in close correlation with some routineously determined semen parameters. These ejaculat parameters are used for quality classification and selection of ejaculates and are according to long term experience in good correlation to the male fertility. Semen quality related correlations could not be found for the dipeptidyl aminopeptidase (DPIV) and the aminopeptidase A (APA). Origin and possible function of the zinc containing peptidases in the semen are examined and discussed.

[Possible involvement of the renin-angiotensin system in reproduction. II. Occurrence and role in the female reproductive tract]. / Zur möglichen Mitbeteiligung des Renin- Angiotensin-Systems am Fortpflanzungsgeschehen. II. Vorkommen und Rolle im weiblichen Reproduktionstrakt.

Biochemical and immunological studies of the last years reveal the existence of an "ovarian renin-angiotensin system (RAS)". Despite of the low angiotensin-conterting enzyme (ACE) activity in the ovary the follicular fluid is rich in angiotensin II (AII). The detection of AII receptors on cells within maturating follicles proves them as AII targets. Therefore, it is supposed that AII may be involved in the regulation of fundamental processes of follicle maturation and/or corpus luteum formation. Further interesting findings are the high concentration of prorenin in the follicle fluid of women causing an increase of the prorenin blood plasma level at time of ovulation, and a second increase of the blood prorenin concentration in the middle of the luteal phase. With respect to the ACE activity in the ejaculate it is imaginable that the smooth muscle tonus of the uterus and the oviduct could be affected by local generation von AII and/or degradation of bradykinin and thus the transit of the semen may be facilitated. Further systematic research is necessary to bring more light into the physiological context and to replace hypothetical interpretations of the findings by exact knowledge.

[Possible involvement of the renin-angiotensin system in reproduction. III. Occurrence and role in pregnancy]. / Zur möglichen Mitbeteiligung des Renin-Angiotensin-Systems am Fortpflanzungsgeschehen. III. Vorkommen und Rolle bei der Gravidität.

To clarify a possible participation of the renin-angiotensin-system (RAS) in physiological processes of pregnancy, findings are listed about occurrence and variations of RAS components in uterus, placenta, amniotic fluid, fetal membranes, fetus and serum. There exist complete and independent RAS in the respective organs and tissues. During pregnancy concentrations and/or activities of the components change, but in different ways and non-uniforming. The information now available does not yet allow a definite view on the role of RAS during pregnancy and on the correlations to estrogens, progesterone, chorionic gonadotropin, prostaglandins etc. Supposedly RAS may be involved to a high degree in the local regulation of blood flow and the maintenance of fetal vascular homeostasis. Furthermore, there are relations between fetoplacental components of RAS and pregnancy-induced hypertension. Further investigations are necessary. They are expected to offer therapeutic possibilities for influencing pathological processes in which RAS plays a role.

[Possible involvement of the renin-angiotensin system in reproduction. I. Occurrence and role in the male reproductive tract]. / Zur möglichen Mitbeteiligung des Renin-Angiotensin-Systems am Fortpflanzungsgeschehen. I. Vorkommen und Rolle im männlichen Reproduktionstrakt.

With regard to the blood pressure the regulative function of the renin-angiotensin-system (RAS) is well known. Knowledge of the last years is that components of the RAS are available also in organs and excrets of the male reproductive tract. So, the angiotensin-converting-enzyme (ACE) - a key enzyme of the RAS - exists in the testes in an extraordinary high activity exceeding those of the lung tissue. In the ejaculate this so-called "testicular ACE" is associated with sperms, whereas the seminal plasma contains the higher molecular "pulmonary ACE". Because both isoenzymes posses the same catalytic effectiveness, the occurrence of adequate substrates (angiotensin I, bradykinin) and degrading products as effectors (angiotensin II), respectively, is of high importance for the valuation of the physiological relevance of the RAS in the process of fertility. Up to date, the knowledge is still insufficient, partly even contradictory. Therefore at present the regulative function(s) of the RAS in the male reproductive tract can only be expressed as postulates.

[The determination of neutral metalloendopeptidase (enkephalinase A) in biological material]. / Zur Bestimmung von Neutraler Metalloendopeptidase (Enkephalinase A) in biologischem Material.

The simple determination of the Neutral Metalloendopeptidase (NEP, Enkephalinase A) with the known fluorogenic substrate Dansyl-D-Ala-Gly-(pNO2)Phe-Gly is disturbed by high concentrations of the Angiotensin-Converting-Enzyme (ACE). ACE hydrolyzes this substrate too but to a smaller degree. In some tissues and body fluids a further substrate hydrolysis takes place by any indefinite proteases. Finally the enzymatic hydrolysis of the NEP-substrate is inhibited by phosphate ions. A method is proposed for the elimination of this disturbances in the NEP-determination with a phosphate-free buffer using two comparison tests with Lisinopril and o-Phenanthroline. The resulting NEP-activity is calculated very simple thereafter.