Green tea reduces body fat via upregulation of neprilysin.

BACKGROUND/OBJECTIVE: Consumption of green tea has become increasingly popular, particularly because of claimed reduction in body weight. We recently reported that animals with pharmacological inhibition (by candoxatril) or genetic absence of the endopeptidase neprilysin (NEP) develop an obese phenotype. We now investigated the effect of green tea extract (in drinking water) on body weight and body composition and the mediating role of NEP. SUBJECTS/METHODS: To elucidate the role of NEP in mediating the beneficial effects of green tea extract, 'Berlin fat mice' or NEP-deficient mice and their age- and gender-matched wild-type controls received the extract in two different doses (300 or 600 mg kg-1 body weight per day) in the drinking water. RESULTS: In 'Berlin fat mice', 51 days of green tea treatment did not only prevent fat accumulation (control: day 0: 30.5% fat, day 51: 33.1%; NS) but also reduced significant body fat (green tea: day 0: 27.8%, day 51: 20.9%, P<0.01) and body weight below the initial levels. Green tea reduced food intake. This was paralleled by a selective increase in peripheral (in kidney 17%, in intestine 92%), but not central NEP expression and activity, leading to downregulation of orexigens (like galanin and neuropeptide Y (NPY)) known to be physiological substrates of NEP. Consequently, in NEP-knockout mice, green tea extract failed to reduce body fat/weight. CONCLUSIONS: Our data generate experimental proof for the assumed effects of green tea on body weight and the key role for NEP in such process, and thus open a new avenue for the treatment of obesity.

Effects of kinins on mammalian spermatozoa and the impact of peptidolytic enzymes.

Effects of kinins, mainly bradykinin (Bk), and other components of the kallikrein-kinin system on sperm motility and further fertility-related functions have been described repeatedly. However, reported data are in part controversial and the mechanism of kinin effects on sperm motility is not yet understood. In the present report we describe a significant promoting effect of Bk on sperm motility at subnanomolar concentrations. This effect was stabilized and even increased by suppression of Bk hydrolysis in semen samples. As sperm membrane-bound angiotensin-converting enzyme and neutral metalloendopeptidase are mainly involved in Bk hydrolysis, an effective cocktail of enzyme inhibitors promoting the sperm motility consists of phosphoramidon and lisinopril (both at 10-7 m). The effects of Bk on sperm cells are not mediated by the B2 Bk receptor. Using several biochemical, molecular and genetic methods we could not detect any Bk receptor on spermatozoa.

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.

Inhibition of corticotropin releasing factor (CRF)-induced adrenocorticotropin (ACTH) secretion in pituitary corticotropic cells by substance P.

Substance P (SP) is one of the three distinct peptides of tachykinin system which possess a common spectrum of biological activities including a modulation of stress. It is assumed that the anterior pituitary is one possible target of SP in attenuation the stress response. Therefore the interaction between the hypothalamic stress hormone corticotropin releasing factor (CRF) and SP was investigated in AtT20/D16v-cells, a cellular model derived from a pituitary tumor. CRF stimulates the release of ACTH from AtT20/D16v cells in a concentration dependent manner. SP (1 microM) was able to abolish the CRF (100 nM)-induced ACTH release. In the same way SP inhibited the CRF-induced accumulation of cyclic adenosine monophosphate (cAMP), indicating that SP influenced the signal transduction pathway of CRF receptor activation. Thus, a direct inhibition of the CRF-mediated stress response by SP at the level of anterior pituitary seems to be likely.

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.

Expression and characterization of the substance P (NK1) receptor in the rat pituitary and AtT20 mouse pituitary tumor cells.

Although substance P is known to take part in the regulation of the anterior pituitary, no conclusive evidence for the expression of the tachykinin NK1 receptor has been found yet in the pituitary or pituitary derived cells. With the reverse transcription-polymerase chain reaction (RT-PCR) method we could detect the low abundant transcripts of the NK1 receptor in the rat pituitary and in the AtT20 cell line (clone D16v). Furthermore, the functional expression of the NK1 receptor in AtT20 cells was confirmed by activation of the phosphatidylinositol-calcium second messenger system when the cells were treated with substance P. In addition, binding studies also indicated the functional expression of this receptor in AtT20 cells. Thus we provide the first evidence that the NK1 receptor is expressed in AtT20 cells and the rat pituitary.

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.

[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.

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.

[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.

Is dipyridamole an angiogenic agent?

Dipyridamole, a potent inhibitor of the nucleoside transport into cells, has recently been reported to stimulate the proliferation of capillary endothelial cells in the heart and skeletal muscle of rats following long-term treatment. We tested this drug on cultured calf aortic endothelial cells and obtained no evidence that dipyridamole is able to stimulate directly the proliferation and migration of these cells. By using the chorioallantoic membrane model we observed that the pharmaceutical preparation Curantyl induces vascular responses almost regularly. This effect, however, was found to be dependent on the high concentration of hydrogen ions in this preparation and could be prevented by increasing the pH value. A direct angiogenic effect of dipyridamole could not be demonstrated on the CAM.