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.

Gastrointestinal hemorrhage in hemodialysis patients.

INTRODUCTION: Gastrointestinal bleedings are frequently dramatic events. So far hardly any data concerning the frequency of such bleedings in patients treated with chronic hemodialysis are available. Thus, the objective of this study is to collect data about the incidence of such events in a dialysis center. MATERIALS AND METHODS: Data of a total of 65 patients who had been treated in the dialysis center between January 01, 2000 and February 28, 2007, including all episodes of gastrointestinal bleedings, have been collected and a correlation with the duration of treatment with hemodialysis has been established. Afterwards we differentiated between those patients who additionally were treated with anticoagulants and those who were not. RESULTS: In a total of 3195 months of treatment, 11 gastrointestinal bleedings were observed. In patients who underwent anticoagulation therapy the risk of experiencing gastrointestinal bleeding was approximately 3 times higher (one episode of bleeding in 16 years) than in the other patients (one episode of bleeding in 46.2 years). DISCUSSION: Compared to the general population, the incidence of gastrointestinal bleeding is increased in hemodialysis patients. Treatment with anticoagulants leads to an even higher risk. Consequently, with regard to concomitant medication which might contribute to increasing the risk of gastrointestinal bleeding, the necessity and therapeutic benefits of the treatment have to be carefully weighed against the potential risks that it might carry for the individual patient.

Continuous ambulatory peritoneal dialysis--developments during changing times.

INTRODUCTION: Continuous ambulatory peritoneal dialysis (CAPD) treatment is subject to constant changes. Departing from this thesis, it should be analyzed in which respects CAPD treatment at the Heidelberg outpatient clinic has changed when considering demographic data, and CAPD-specific data including infection rates and clinical parameters. MATERIALS AND METHODS: A retrospective study was carried out in the course of which a cohort of 67 CAPD patients treated before the year 2000 was compared with a cohort of 53 patients treated from the year 2000 on. Demographic data, CAPD-specific and clinical data at the commencement of treatment and data gathered during the observation period thereafter were recorded. RESULTS: The patients of the more recent cohort were 5 years older. At the initiation of treatment, the volume of residual diuresis was higher in the patients treated from 2000 on than in those treated before. The blood pressure in patients belonging to the more recently treated group was also lower than the blood pressure of those treated before. The incidence of peritonitis and exit-site infections has decreased. On the other hand, the hemoglobin level has risen. DISCUSSION: Compared to the past, many parameters have improved. Above all, the findings suggest that medical care and treatment have become better, probably as a result of the modified guidelines. The significant decrease in infections is probably also due to the use of the new dialysis fluids.

Achieving signalling selectivity of ligands for the corticotropin-releasing factor type 1 receptor by modifying the agonist's signalling domain.

BACKGROUND AND PURPOSE: Most of the pharmaceuticals target G-protein-coupled receptors (GPCRs) which can generally activate different signalling events. The aim of this study was to achieve functional selectivity of corticotropin-releasing factor receptor type 1 (CRF(1)) ligands. EXPERIMENTAL APPROACH: We systematically substituted urocortin, a natural peptide agonist of CRF(1), with bulky amino acids (benzoyl-phenylalanine, naphthylalanine) and determined the effect of the analogues on coupling of CRF(1) to Gs- and Gi-protein in human embryonic kidney cells, using receptor binding, [(35)S]-GTPgammaS binding stimulation, and cAMP accumulation assays. KEY RESULTS: Native ligands stimulated Gs and Gi activation through CRF(1), resulting in stimulation and then inhibition of cAMP accumulation. Single replacements in urocortin at positions 6-15 led, dependent on the position and nature of the substituent, to ligands that conserved Gs activity, but were devoid of Gi activity, only stimulating cAMP accumulation, and competitively antagonized the Gi activation by sauvagine. In contrast, analogues with substitutions outside this sequence non-selectively activated Gs and Gi, as urocortin did. CONCLUSIONS AND IMPLICATIONS: Modifications in a specific region, which we have called the signalling domain, in the polypeptide agonist urocortin resulted in analogues that behaved as agonists and, at the same time, antagonists for the activation of different G-proteins by CRF(1). This finding implies significant differences between active conformations of the receptor when coupled to different G-proteins. A similar structural encoding of signalling information in other polypeptide hormone receptor ligands would result in a general concept for the development of signalling-selective drug candidates.

Evidence for downregulation of the endothelin-B-receptor by the use of fluorescent endothelin-1 and a fusion protein consisting of the endothelin-B-receptor and the green fluorescent protein.

We generated fusion proteins consisting of the endothelin-B (ET(B))-receptor and the enhanced green fluorescent protein (EGFP) to visualize receptor internalization. In Madin Darby canine kidney (MDCK) clones expressing ET(B)/EGFP fusion proteins, single class high affinity binding sites for [125I]endothelin-1 (ET-1) were found (for two different clones apparent K(D) values were 31 +/- 15 pM and 30 +/- 7 pM). Pretreatment of membranes with GTPgammaS prior to saturation analysis did not alter these values. We also labelled ET-1 with cyanine-dyes (Cy3/ET-1, Cy5/ET-1). In displacement analyses with membranes of MDCK ET(B)/EGFP clones using [125I]ET-1, we found reduced affinity for Cy3/ET-1 and Cy5/ET-1 (about 5- to 10-fold, respectively), but normal efficacy when compared to unlabelled ET-1. Both fluorescent ligands and the ET(B)/EGFP fusion protein were suitable for analysis of receptor trafficking in living cells and cells fixed at different timepoints. Laser scanning microscopy of MDCK ET(B)/EGFP clones incubated with Cy3/ET-1 or Cy5/ET-1 revealed rapid internalization of ligand/receptor complexes, which clustered in large, perinuclear structures (most probably late endosomes). Our data argue against recycling of the ET(B) receptor and favour its targeting to the lysosomal pathway.

Late endosomal/lysosomal targeting and lack of recycling of the ligand-occupied endothelin B receptor.

A fusion protein consisting of the endothelin B (ET(B)) receptor and the enhanced green fluorescent protein (EGFP) in conjunction with Cyanin3- or fluorescein-conjugated endothelin 1 (Cy3-ET1, Fluo-ET1) was used to investigate the ligand-mediated internalization of the ET(B) receptor. The ET(B) receptor and the ET(B)/EGFP fusion protein displayed very similar pharmacological properties when expressed in Chinese hamster ovary cells. The integrity of the fusion protein was verified by low temperature PAGE analysis of the (125)I-ET1-bound ET(B) receptor and the (125)I-ET1-bound ET(B)/EGFP fusion protein. Fluorescence microscopy of Chinese hamster ovary cells expressing the ET(B)/EGFP fusion protein demonstrated strong signals at the plasma membrane. On addition of Cy3-ET1, internalization of ligand and receptor occurred within 5 min via a sucrose-sensitive (i.e., clathrin-mediated) pathway. On further incubation, ET(B)/EGFP and Cy3-ET1 fluorescences were found in the perinuclear region, colocalized with fluorescent low density lipoproteins, a marker of the late endosomal/lysosomal pathway, but not with fluorescent transferrin, a marker of the recycling pathway. No dissociation of Cy3-ET1 from the receptor was seen within 4 h. Using (125)I-ET1 or Cy3-ET1, binding sites were again demonstrable at the cell surface within 2 h. The reappearance of binding sites was abolished by prior treatment of the cells with cycloheximide, an inhibitor of protein synthesis. The data demonstrate that the ligand-occupied ET(B) receptor is internalized; however, it does not recycle like most of the G protein-coupled receptors but is sorted to the late endosomal/lysosomal pathway in a manner similar to that of the family of protease-activated receptors.

A role for a helical connector between two receptor binding sites of a long-chain peptide hormone.

The conformational freedom of single-chain peptide hormones, such as the 41-amino acid hormone corticotropin releasing factor (CRF), is a major obstacle to the determination of their biologically relevant conformation, and thus hampers insights into the mechanism of ligand-receptor interaction. Since N- and C-terminal truncations of CRF lead to loss of biological activity, it has been thought that almost the entire peptide is essential for receptor activation. Here we show the existence of two segregated receptor binding sites at the N and C termini of CRF, connection of which is essential for receptor binding and activation. Connection of the two binding sites by highly flexible epsilon-aminocaproic acid residues resulted in CRF analogues that remained full, although weak agonists (EC(50): 100-300 nM) independent of linker length. Connection of the two sites by an appropriate helical peptide led to a very potent analogue, which adopted, in contrast to CRF itself, a stable, monomer conformation in aqueous solution. Analogues in which the two sites were connected by helical linkers of different lengths were potent agonists; their significantly different biopotencies (EC(50): 0.6-50 nM), however, suggest the relative orientation between the two binding sites rather than the maintenance of a distinct distance between them to be essential for a high potency.

The role of conserved extracellular cysteine residues in vasopressin V2 receptor function and properties of two naturally occurring mutant receptors with additional extracellular cysteine residues.

The G protein-coupled vasopressin V2 receptor (V2 receptor) contains a pair of conserved cysteine residues (C112 and C192) which are thought to form a disulfide bond between the first and second extracellular loops. The conserved cysteine residues were found to be important for the correct formation of the ligand binding domain of some G protein-coupled receptors. Here we have assessed the properties of the V2 receptor after site-directed mutagenesis of its conserved cysteine residues in transiently transfected human embryonic kidney (HEK 293) cells. Mutant receptors (C112S, C112A and C192S, C192A) were non-functional and located mostly in the cell's interior. The conserved cysteine residues of the V2 receptor are thus not only important for the structure of the ligand binding domain but also for efficient intracellular receptor transport. In addition to the functional significance of the conserved cysteine residues, we have also analyzed the defects of two mutant V2 receptors which cause X-linked nephrogenic diabetes insipidus (NDI) by the introduction of additional cysteine residues into the second extracellular loop (mutants G185C, R202C). These mutations are assumed to impair normal disulfide bond formation. Mutant receptor G185C and R202C were efficiently transported to the plasma membrane but were defective in ligand binding. Only in the case of the mutant receptor R202C, the more sensitive adenylyl cyclase activity assay revealed vasopressin-stimulated cAMP formation with a 35-fold increased EC(50) value and with a reduced EC(max), indicating that ligand binding is not completely abolished. Taking the unaffected intracellular transport of both NDI-causing mutant receptors into account, our results indicate that the observed impairment of ligand binding by the additional cysteine residues is not due to the prevention of disulfide bond formation between the conserved cysteine residues.

Cutaneous expression of CRH and CRH-R. Is there a "skin stress response system?".

The classical neuroendocrine pathway for response to systemic stress is by hypothalamic release of corticotropin releasing hormone (CRH), subsequent activation of pituitary CRH receptors (CRH-R), and production and release of proopiomelanocortin (POMC) derived peptides. It has been proposed that an equivalent to the hypothalamic-pituitary-adrenal axis functions in mammalian skin, in response to local stress (see Reference 1). To further define such system we used immunocytochemistry, RP-HPLC separation, and RIA techniques, in rodent and human skin, and in cultured normal and malignant melanocytes and keratinocytes. Production of mRNA for CRH-R1 was documented in mouse and human skin using RT-PCR and Northern blot techniques; CRH binding sites and CRH-R1 protein were also identified. Addition of CRH to immortalized human keratinocytes, and to rodent and human melanoma cells induced rapid, specific, and dose-dependent increases in intracellular Ca2+. The latter were inhibited by the CRH antagonist alpha-helical-CRH(9-41) and by the depletion of extracellular calcium with EGTA. CRH production was enhanced by ultraviolet light radiation and forskolin (a stimulator for intracellular cAMP production), and inhibited by dexamethasone. Thus, evidence that skin cells, both produce CRH and express functional CRH-R1, supports the existence of a local CRH/CRH-R neuroendocrine pathway that may be activated within the context of a skin stress response system.

Adaptation of corticosterone-but not beta-endorphin-secretion to repeated blood sampling in rats.

Effects of short-term repeated blood sampling on the secretion of corticosterone (CORT) and beta-endorphin (beta-END) were evaluated in male Wistar rats. Blood was drawn from the tail vein of conscious rats four times within 2 h both at the peak and trough period of the diurnal corticosterone secretion cycle. All rats were well accustomed to the procedure. The main findings were: (1) At both sampling intervals, CORT increased significantly in response to the first sampling and declined to baseline values in successive samples. (2) beta-END also increased significantly in response to the first sampling but remained elevated in successive samples. (3) Intensities of initial CORT and beta-END responses correlated positively with each other and with the baseline beta-END values. Feedback inhibition of CORT secretion with sustained elevation of beta-END titres suggests a moderate stress intensity of the repeated blood sampling procedures. In general, due to lack of short-term feedback inhibition, beta-END seems to reflect the effects of repeated administration of moderate intense stressors more closely than CORT.

The degradation of corticotropin-releasing factor by enzymes of the rat brain studied by liquid chromatography-mass spectrometry.

The corticotropin-releasing factor (CRF; 41 amino acid residues) is a major regulatory peptide in the response to stress and is distributed over many regions of the brain. We have studied the enzymatic degradation of CRF and related peptides by the CRF-degrading enzyme(s) of the rat brain (CRF-DA) by liquid-chromatographic-mass spectrometric technique and by online tandem mass spectrometric experiments. Peptide fragments of the human/rat CRF (1-41) generated by the CRF-DA of the particulate cell fraction were separated and structurally assigned. Major sites of enzymatic attack were identified at the P1 positions Ser1, Thr11 , His13, Leu15, Arg23, Arg35, and Lys36 with Leu15 as the site of primary cleavage. The CRF-DA was shown to be dominated by a metalloendopeptidase activity inhibited by O-phenanthroline and EDTA. The cytosolic fraction generated a similar degradation pattern with a pronounced cleavage at the Arg35 position.

Hair cycle-dependent expression of corticotropin-releasing factor (CRF) and CRF receptors in murine skin.

We demonstrate the presence and hair cycle-dependent expression of corticotropin-releasing factor (CRF) and CRF receptors (CRF-R) in C57BL/6 mouse skin. To correlate this with a physiological, developmentally controlled tissue remodeling process, we have analyzed CRF and CRF-R expression during defined stages of the murine hair cycle with its rhythmic changes between growth (anagen), regression (catagen), and resting (telogen). Using reversed-phase HPLC combined with two independent anti-CRF radioimmunoassays, we have identified CRF in murine skin. Maximal CRF levels were found in anagen III-IV skin, and minimal values were detected in catagen and telogen skin. By immunofluorescence, maximal CRF immunoreactivity (CRF-IR) was seen in the basal epidermis, nerve bundles of skin, the outer root sheath and matrix region of anagen IV-VI follicles, and in defined sections of their perifollicular neural network, whereas catagen and telogen skin displayed minimal CRF-IR. Using quantitative autoradiography and 125I-CRF as a tracer, high-affinity binding sites for CRF were detected in murine skin. The highest density of specific binding sites was detected in the panniculus carnosus, the epidermis, and the hair follicle. CRF-R type 1 (CRF-R1) IR was detected by immunohistology mainly in the outer root sheath, hair matrix, and dermal papilla of anagen VI follicles, as well as in the inner and outer root sheaths of early catagen follicles. CRF-R1 expression was also hair cycle dependent. Therefore, in normal murine skin, the CRF-CRF-R signaling system may operate as an additional neuroendocrine pathway regulating skin functions, possibly in the context of cutaneous stress responses.

Corticotropin-releasing factor (CRF) agonists stimulate testosterone production in mouse leydig cells through CRF receptor-1.

The influence of CRF on testosterone production in primary mouse Leydig cell cultures was studied, and the type of CRF receptor (CRF-R) involved in this activity was determined. CRF directly stimulated testosterone production in mouse Leydig cells, but did not influence the maximum human (h)CG-induced testosterone production. The effect was time- and dose-dependent, saturable with an EC50 of 2.84 nM for hCRF, antagonized by the CRF antagonist alpha-helical CRF9-41, and accompanied by intracellular cAMP elevation. The rank order of potency of the natural CRF agonists, hCRF, ovine CRF, sauvagine, and urotensin, corresponded to that of their activities on CRF-R1 in rat pituitary cells and also to that reported for this receptor, but not for CRF-R2, when transfected into various cell lines. Furthermore, the difference in response of mouse Leydig cells to [11-D-Thr,12-D-Phe]- and [13-D-His,14-D-Leu]-ovine CRF corresponded to that measured when COS cells expressing CRF-R1 were activated, but was considerably smaller than that observed for activation of COS cells expressing CRF-R2alpha or -R2beta. The messenger RNA encoding the mouse CRF-R1 was detected by RT-PCR in mouse Leydig cell preparations. In contrast to mouse Leydig cells, CRF agonists had no influence on the basal testosterone and cAMP production by rat Leydig cells, nor did the agonists or antagonist change the hCG-stimulated testosterone and cAMP production by these cells. It is concluded that mouse Leydig cells express CRF-R1, mediating elevation of testosterone production by CRF agonists through cAMP. Because potencies of CRF agonists in activating mouse Leydig cells were more than 10-fold lower compared with their potencies in stimulating rat pituitary cells, it is suggested that the coupling of the CRF-R1 to intracellular signaling in Leydig cells is different from that in corticotropic pituitary cells, at least in quantitative terms.

Identification and measurement of beta-endorphin levels in the skin during induced hair growth in mice.

We describe new and effective techniques for extracting proopiomelanocortin (POMC)-derived peptides from mammaliar skin. Using this methodology (hot-acid extraction) and two independent HPLC-controlled RIA systems, we identify beta-endorphin peptide in mammalian skin and demonstrate significant hair cycle-dependent fluctuations in both the skin concentration and the in situ expression pattern of beta-endorphin (sebaceous glands) during the entire murine hair cycle. The observed anagen (growth phase) associated increase in beta-endorphin concentration and its decline during the follicle involution (catagen) or resting (telogen) phase raise the possibility of a regulatory function of this neuropeptide in cyclic changes of skin physiology.

Corticotropin-releasing hormone (CRH) receptors in the mesenteric small arteries of rats resemble the (2)-subtype.

The potencies of the corticotropin-releasing hormone (CRH) agonistic peptides oCRH, h/rCRH, frog sauvagine, and carp urotensin I and of the antagonistic peptide alpha-helical CRH9-41 were compared in 3 different in vitro assays: (a) receptor binding to rat brain membranes; (b) release of ACTH/beta-endorphin from rat pituitary cells; and (c) relaxation of rat mesenteric small arteries. From their potency profiles, especially from the high potency of sauvagine relative to CRH in the relaxation assay, it is concluded that the receptors mediating the hypotensive action of systemic CRH in vascular smooth muscle are different from those in the pituitary and brain, and may be identical or very similar to the recently cloned new CRH receptor type 2.

A single-point slight alteration set as a tool for structure-activity relationship studies of ovine corticotropin releasing factor.

In order to determine which amino acid side chains of ovine corticotropin releasing factor (oCRF) are most sensitive to alterations with respect to receptor binding and activation, we synthesized a single-point replacement set by replacing each residue by a similar, preferably proteinogenic amino acid, maintaining a minimal change of character at each position (Ser by Thr, Gln by Asn, Glu by Asp, Arg by Lys, and vice versa, Pro by N-MeAla, Ile by Leu, Leu by Nle, Phe by Trp, His by Ala, Val by Leu, Met by Nle, Ala by Leu). In general, any loss in the biological potency by a single-point substitution in oCRF parallels a decrease in receptor binding, indicating that, in contrast to previous suggestions, there is no specific side chain in the peptide that is more responsible for receptor activation than for receptor binding. In addition to Arg(16), Ala(31), and Arg(35), amino acid residues in the N-terminal sequence (5-14) were found to be sensitive to alteration, demonstrating their particular importance for the receptor interaction of CRF agonists. Most of the analogs tested exhibited agonistic potencies in an in vitro pituitary cell culture assay at a concentration of 0.3 nM, and all analogs showed full agonistic potency at 1 microM. In contrast to the results of an alanine replacement study, the strongest decrease in receptor binding and biological potency was observed for analogs with substitutions of hydrophilic amino acids Ser(7), Arg(16), Glu(17), or Asn(34). In the case of Ser(7) and Arg(16), side chain specific interactions with the receptor may be required for high affinity. Alanine replacements at positions 17 or 34 resulted in analogs that were as potent as oCRF, while replacement of Glu(17) by Asp or Asn(34) by Gln caused a dramatic loss in potency, thereby suggesting an important effect at sterically or conformationally sensitive positions. In contrast to corresponding alanine analogs which exhibited a significant loss in biological potency, slight alterations of lipophilic side chains at positions 6, 12, or 38 did not cause a significant reduction of receptor binding and activation, indicating that it is not specific side chains but rather lipophilicity which is essential at these positions. Indeed, replacement of Phe(12) by Trp provides an agonist with significantly increased receptor binding and biological potency.

Determination of substance P in human nasal lavage fluid.

The determination of substance P (SP) concentrations in human nasal lavages can be used to monitor physiological and certain pathophysiological processes in human airway mucosa. But, because of the low concentrations, immunoassays of high sensitivity are needed. Two approaches to improve the sensitivity of the radioimmunological determinations of SP are compared: increasing the sample volume and miniaturizing the assay design. The characterization of SP-like immunoreactivity (SP-LIR) in human nasal lavage was performed by investigating the immunological specificity of the antibody used in the radioimmunoassays and by reversed-phase high-performance liquid chromatography separation of the SP-LIR. SP concentrations in nasal lavages can be reliably measured by each of the two introduced RIA methods. Despite the lower detection limit of the miniaturized immunoassay (0.2 in comparison to 1.3 fmol/incubate) it is advisable to increase the sample volume in order to improve the sensitivity because of the higher precision of the determinations. SP-LIR was found in nasal lavage specimens in concentrations between 2 and 10 fmol/ml and consisted of authentic SP and, to a less extent, SP-sulfoxide.

Effects of voluntary ethanol ingestion on the POMC gene expression in the rat pituitary and on the plasma beta-endorphin content.

Studies investigating the influence of chronic ethanol treatment on the beta-endorphin content and the proopiomelanocortin (POMC) gene expression in the rat pituitary revealed contradictory results. Because of this we decided to start a more complex study to investigate the effects of isolation stress, chronic ethanol treatment and voluntary ethanol consumption on the POMC mRNA level in the rat pituitary. The immediately prepared total RNA from rat pituitaries was used in hybridization experiments (Northern- and Dot-blots). The results suggest a correlation between the POMC gene expression and the different fashions of 'living conditions' tested. So the POMC gene expression in long-term alcohol-treated animals was decreased supporting the theory of beta-endorphin deficiency in alcoholism. More interestingly, data obtained from the group of voluntary ethanol consumption suggest an inverse correlation between total ethanol ingestion and POMC gene expression. This indicates the importance of the method of ethanol administration. Consistent with a decreased POMC gene expression in the pituitary during chronic ethanol treatment are previous studies showing a decrease in the plasma beta-endorphin content in such situations. Surprisingly, in the present study the plasma beta-endorphin levels measured by radioimmunoassay were only slightly decreased in chronically ethanol-treated rats. This may be due to dysregulatory effects of ethanol on post-translational processing, degradation and/or release of beta-endorphin.