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A full-length cDNA clone coding for rat napsin was identified by homology search of the ZooSeq rat EST database (Incyte). Northern blot analysis revealed high expression of napsin mRNA transcripts in kidney, lung and spleen. Western blot analysis showed that rat napsin is expressed in kidney as a 50-kDa, highly glycosylated, monomeric protein. Lysates prepared from human embryonic kidney cells (HEK293) transfected with rat napsin showed increased enzymatic activity which was inhibited by pepstatin.

The identification, synthesis and activity of a novel class of piperidine renin inhibitors is presented. The most active compounds show activities in the picomolar range and are among the most potent renin inhibitors ever identified.

The cloning of a cDNA encoding a G protein-coupled receptor homologous to the endothelin type B receptor, but unable to bind endothelin, was recently reported and termed ETBR-LP. We report here the isolation of a human cDNA encoding a receptor that is highly related to ETBR-LP and which was therefore termed ETBR-LP-2. Comparison of the two amino acid sequences revealed 68% overall homology and 48% identity. As is the case for ETBR-LP, the new receptor is strongly expressed in the human central nervous system (e.g. in cerebellar Bergmann glia, cerebral cortex, internal capsule fibers). Membranes of HEK-293 cells stably expressing ETBR-LP-2 did not bind endothelin-1, endothelin-2, endothelin-3, bombesin, cholecystokinin-8 or gastrin-releasing peptide.

Implementation of a pyridylcarbamoyl group and an isopropylpyridylsulfonamide substituent as key components in the scaffold of Bosentan resulted in the identification of the potent orally active endothelin receptor antagonist Ro 48-5695. It shows affinities for ETA and ETB receptors in the low nanomolar range and high functional antagonistic potency in vitro.

We have identified the human counterpart of the corticotropin-releasing factor receptor subtype 2beta. Its functional response to human urocortin was demonstrated after stable expression in HEK-293 cells. The receptor was also shown to bind sauvagine, corticotropin-releasing factor and urocortin. In contrast to rodents, the human CRF(2beta) receptor is only weakly expressed in heart and skeletal tissues, where the CRF(2alpha) isoform is predominant. Moreover, we have identified additional mRNAs of the CRF(2beta) type which are probably a consequence of aberrant splicing events.

Human renin has been expressed in Sf9 and CHO cells using two different gene constructs. The first construct contained a foreign signal peptide fused directly to the sequence encoding mature renin, whereas the second construct harbors the sequence for preprorenin. Prorenin was produced in significantly higher amounts than the mature enzyme expressed without its propeptide in both expression systems. Both directly expressed mature renin and proteolytically derived active renin have been purified and cocrystallized with the renin inhibitor Ro 42-5892. The 3D structure has been solved for both versions and demonstrates identity despite different glycosylation and different N termini.

A three-dimensional model for the transmembrane domains of human endothelin-A receptor was built using structural information from bacteriorhodopsin and sequence alignment to other guanine-nucleotide-binding regulatory(G) protein-coupled receptors. Based on this model, 18 amino acids located at the inside of the receptor were mutated and analyzed for binding of the natural ligand endothelin-1 and bosentan, a recently described potent orally active endothelin antagonist [Clozel, M., Breu, V., Gray, G., Kalina, B., Loffler, B.-M., Burri, K., Cassal, J.-M., Hirth, G., Muller, M., Neidhart, W. & Ramuz, H. (1994) Pharmacological characterization of bosentan, a new potent orally active nonpeptide endothelin receptor antagonist, J. Pharmacol. Exp. Ther. 270, 228-235]. Mutation of Gly97, Lys140, Lys159, Gln165 and Phe315, located in transmembrane region 1, 2, 3, 3, and 6, respectively, caused reduced specific binding of 125I-labelled endothelin-1, despite an expression level similar to wild-type endothelin-A receptor. Mutation of Tyr263, Arg326 and Asp351 preserved endothelin-1 binding but caused reduced binding of bosentan. These amino acids, located on transmembrane regions 5, 6 and 7, respectively, are conserved among endothelin-A and endothelin-B receptors but not in other G-protein-coupled receptors. These observations demonstrate a dissociation of the binding site for the peptidic natural agonist endothelin-1 and the synthetic non-peptide antagonist bosentan. They provide the molecular basis for bosentan being a specific antagonist for both, endothelin-A as well as endothelin-B receptors and may in combination with studies on structure/activity relationship support the design of novel and more potent endothelin receptor antagonists.

SPECIFIC AIMSAspirin inhibits IKKβ in vitro; however, the in vivo relevance of the phenomenon is unclear. We have developed a model of severe angiotensin (Ang) II-induced vascular injury and now tested the hypothesis that chronic aspirin treatment of transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR) protects from Ang II-induced end organ damage by inhibiting NF-κB activation in vivo.PRINCIPAL FINDINGSHigh-dose aspirin reduces mortality and renal damage independent of blood pressureRenal glomerulus of untreated 7-wk-old dTGR shows mesangial expansion and podocyte damage. Renal dTGR arterioles exhibit severe changes in wall structure, including proliferation of smooth muscle cells and deposition of dense vacuoles. In contrast, nontransgenic Sprague-Dawley (SD) rats showed no vascular or glomerular damage. Untreated dTGR show signs of glomerular sclerosis and vascular damage with mortality > 50% at wk 7. High-dose aspirin reduced mortality to < 10% whereas low-dose aspirin produ...

It has been suggested that the endothelin (ET) ETB receptor could mediate endothelium-dependent vasodilation to ET-1 or ET-3, but its in vivo role is still largely unknown. We used sarafotoxin S6C, a selective agonist of the ETB receptor, to study the in vivo effects of ETB stimulation. SRTX S6C induced a transient decrease in blood pressure, followed by a long-lasting pressor response accompanied by a marked renal and mesenteric vasoconstriction. No constriction was observed in isolated mesenteric arteries in vitro, indicating that the in vivo vasoconstrictor effect is most likely indirect. The pressor effect of SRTX S6C was not dependent on central stimulation of ETB receptors and was not mediated by catecholamines from the adrenal medulla, prostanoids or ET-1.

The goal of our study was to evaluate and compare the effects of receptor blockade with different endothelin (ET) receptor antagonists on plasma concentrations of ET-1, big ET-1 and ET-3 in conscious rats. Ro 46-2005 (10 mgkg, i.v.), a novel non-peptide antagonist of both ETA and ETB receptors, increased the concentrations of ET-1 in plasma to 200 ± 13% of basal levels (P < 0.001). This effect was dose- and time-dependent and reached a maximum at 15 min. Ro 46-2005 had no effect on plasma concentrations of big ET-1 and only a minor effect on those of ET-3. In contrast to Ro 46-2005, the selective peptide ETA antagonists BQ-123 and FR-139317 had no effect on plasma ET-1 concentrations. The increase in plasma ET-1 concentrations by Ro 46-2005 was most likely not due to de novo synthesis, since big ET-1 levels were not increased and peak levels were reached early after compound injection, but perhaps to displacement of ET-1 from the ETB receptors.

Non-peptidomimetic renin inhibitors of the piperidine type represent a novel structural class of compounds potentially free of the drawbacks seen with peptidomimetic compounds so far. Synthetic optimization in two structural series focusing on improvement of potency, as well as on physicochemical properties and metabolic stability, has led to the identification of two candidate compounds 14 and 23. Both display potent and long-lasting blood pressure lowering effects in conscious sodium-depleted marmoset monkeys and double transgenic rats harboring both the human angiotensinogen and the human renin genes. In addition, 14 normalizes albuminuria and kidney tissue damage in these rats when given over a period of 4 weeks. These data suggest that treatment of chronic renal failure patients with a renin inhibitor might result in a significant improvement of the disease status.

The poor interspecies conservation of the renin-angiotensin system prevents the use of nonprimate in vivo models to test renin inhibitors. Thus the small New-World monkey marmoset is used in many instances as a model. However, large differences between the potencies of renin inhibitors as measured in human and marmoset plasma were observed. To understand this phenomenon, we cloned marmoset renin and angiotensinogen. They were highly homologous to their human counterparts, except for a six-residue deletion in the marmoset renin propeptide. Human and marmoset recombinant renins were found in vitro to display comparable activities, suggesting that the observed differences in plasma apparent affinity of inhibitors could be due to different plasma protein binding of the inhibitors.

Piperidine renin inhibitors with heterocyclic core modifications or hydrophilic attachments show improved physical properties (lower lipophilicity, improved solubility). Tetrahydroquinoline derivative rac-30 with a molecular weight of 517 and a log D(pH 7.4) of 1.9 displays potent and long lasting blood pressure lowering effects after oral administration to sodium depleted conscious marmosets.

Background The aspartic proteinase renin catalyses the first and rate-limiting step in the conversion of angiotensinogen to the hormone angiotensin II, and therefore plays an important physiological role in the regulation of blood pressure. Numerous potent peptidomimetic inhibitors of this important drug target have been developed, but none of these compounds have progressed past clinical phase II trials. Limited oral bioavailability or excessive production costs have prevented these inhibitors from becoming new antihypertensive drugs. We were interested in developing new nonpeptidomimetic renin inhibitors. Results High-throughput screening of the Roche compound library identified a simple 3,4-disubstituted piperidine lead compound. We determined the crystal structures of recombinant human renin complexed with two representatives of this new class. Binding of these substituted piperidine derivatives is accompanied by major induced-fit adaptations around the enzyme's active site. Conclusions The efficient optimisation of the piperidine inhibitors was facilitated by structural analysis of the renin active site in two renin-inhibitor complexes (some of the piperidine derivatives have picomolar affinities for renin). These structural changes provide the basis for a novel paradigm for inhibition of monomeric aspartic proteinases.

Glutamate 1-semialdehyde aminotransferase has been separated from metabolically related activities by gel filtration and affinity chromatography. The enzyme was inhibited by gabaculin, 4-amino 5-fluoropentanoic acid and pyridoxal 5-phosphate and stimulated by pyridoxamine 5-phosphate. The activity of enzyme recovered by elution after electrophoresis in non-denaturing polyacrylamide gels was wholly dependent on pyridoxamine 5-phosphate. A mechanism for the enzyme-catalysed reaction based on these observations is discussed.

The nature of the intermediates of the C5-pathway has been a matter of controversy for a long time. Although the involvement of glutamate-1-semialdehyde (G-l-SA) (1) is widely accepted, the role of 4,5-dioxovalerate (DOVA) (2) as a catalytic component in the C5-pathway is still challenged (3). Well established is yet the activation of glutamate for the formation of 5-aminolevulinate (ALA) by a tRNAglu as demonstrated for barley (4), Chlamydomonas (5), Chlorella (6), Methanobacterium (7), Euglena (8), Cyanidium (9), Synechocystis (9) and spinach (9).

In plants, algae, cyanobacteria and some other bacterial species 5-aminolevulinate (ALA) is synthesized from glutamate (1–6). This reaction sequence involves ligation of glutamate to a tRNAGlu species and subsequent reduction to glutamate-1-semialdehyde (GSA) (7). The conversion of GSA to ALA is then catalysed by GSA-aminotransferase (E.C. 5.4.3.8.) (2,8). Dependent on the requirement of pyridoxamine-phosphate (PAMP) or pyridoxal-phosphate (PALP) for activity, either 4,5-diaminovalerate (8–10) or 4,5-dioxovalerate (DOVA) (5,11,12) are proposed as intermediates.

Besides the known role of 4,5-dioxovalerate as an intermediate in the C 5 -pathway [1], a novel regulatory function in light-dependent chlorophyll biosynthesis is described. Considerable amounts of protochlorophyllide accumulate in dark grown cultures of the yellow mutant C-2A′ of Scenedesmus [2]. This accumulation is almost completely blocked in darkness by the addition of 4,5-dioxovalerate in vivo. Likewise, light-dependent chlorophyll biosynthesis is strongly inhibited by the addition of this compound during greening. The considerable increase of protochlorophyllide formation in darkness upon the addition of 5-aminolevulinate (Kotzabasis, K. and Senger, H. (1989) Z. Naturforsch., in press) is also drastically reduced by external 4,5-dioxovalerate. It is shown by in vitro experiments that concentrations of dioxovalerate, above the physiologically relevant level, inhibit 5-aminolevulinate dehydratase. The K i -value was determined to be 60±5 μM. From these results it is concluded that besides the predominant control of gli t-RNA-ligase by protochlorophyllide [4] a second regulatory mechanism is involved in chlorophyll biosynthesis. Under excessive concentrations of 5-aminolevulinate and 4,5-dioxovalerate further prophobilinogen and subsequently chlorophyll biosynthesis is inhibited.

A small basic 10 kD abundant protein in barley seeds can convey up to 7% of the phosphatidylcholine in liposomes to potato mitochondria whereas cholesteryloleate is not transported. The demonstration of this activity combined with a somewhat more than 50% homology of its primary structure to that of other plant phospholipid transfer proteins are the bases for our naming it a barley lipid transfer protein (LTP).

The involvement of the C5-pathway in the biosynthesis of 5-aminolevulinate for the formation of chlorophyll is proven beyond doubt. At least two reaction mechanisms have been proposed for the C5-pathway, which converts glutamate to 5-aminolevulinate: one involves 4,5-dioxovalerate and the other glutamate-1-semialdehyde. Evidence for the intermediates in vivo is generally considered of great importance for the proof of one or the other pathway. However, another favourable role for 4,5-dioxovalerate is as an intermediate in the conversion of glutamate-1-semialdehyde to 5-aminolevulinate. A recent report that 4,5-dioxovalerate is only an artefact formed duriing the assay [1] contradicts earlier results identifying it in extracts from Scenedesmus cells [2,3]. In the present paper the in vivo formation of thic compound is again demonstrated by different methods of isolation and characterization, including TLC and HPLC followed by absorption and fluorescence measurements and mass spectroscopy. The objection that the diaminonaphthalene derivative of 4,5-dioxovalerate is formed exclusively by the condensation of 5-aminolevulinate with 2,3-diaminonaphthalene [1] is disproved in this paper and the formation and existence of 4,5-dioxovalerate in vivo is confirmed.

We describe here Ro 46-8443, the first non-peptide endothelin ETB receptor selective antagonist. It displays up to 2000-fold selectivity for ETB receptors both in terms of binding inhibitory potency and functional inhibition. The observed parallel rightward shift of concentration-response curves with different antagonist concentrations is consistent with a competitive binding mode. Since Ro 46-8443 selectively inhibits ETB receptor mediated responses, it is a valuable tool for clarifying the role of ETB receptors in pathology.

Ro 46-2005 is a new synthetic non-peptide endothelin (ET) receptor antagonist. In binding experiments, Ro 46-2005 proved to be equipotent (IC50 200–500 nM) for inhibition of [125I]ET-1 binding on the two known ET receptor subtypes (ETA and ETB). Scatchard analysis was consistent with a competitive binding mode. Ro 46-2005 also inhibited the functional consequences of ET-1 stimulation: the ET-1-induced release of arachidonic acid from rat mesangial cells was inhibited with an IC50 of 1.8 μM.

We used Ro 46-8443, non-peptidic antagonist selective of endothelin ETB receptors, to study the role of ETB receptors in rat hypertension models. In normotensive rats, Ro 46-8443 decreased blood pressure, but in SHR and DOCA rats, it induced a pressor effect, due to blockade of ETB-mediated release of nitric oxide since L-NAME prevented it. In rats rendered hypertensive by chronic L-NAME, Ro 46-8443 did not induce a pressor but depressor effect. Thus, in DOCA rats and SHR, Ro 46-8443 reveals a predominant influence of endothelial ‘vasorelaxant’ ETB receptors, while in normotensive rats the prevailing role of ETB receptors seems to be in mediating a vasoconstrictor tone.

In the yellow mutant C-2A′ of the unicellular green alga Scenedesmus obliquus the participation of an RNA species in the conversion of glutamate to 5-aminolevulinate is clearly demonstrated by the fact that RNAase treatment of a soluble enzyme preparation drastically decreases the formation of 5-aminolevulinate. The involvement of 4,5-dioxovalerate in the C5 pathway is demonstrated by the decrease of label in enzymatically formed 5-aminolevulinate from [14C]glutamate by providing an increased unlabelled pool of 4,5-dioxovalerate. Evidence supporting the role of glutamate-1-semialdehyde as an additional intermediate in the reaction sequence is also presented. We propose a new reaction scheme, consistent with the results reported here, for the formation of 5-aminolevulinate via the C5 pathway.