Na,K-ATPase inhibitors from bovine hypothalamus and human plasma are different from ouabain: nanogram scale CD structural analysis.

The specific, high affinity binding of plant-derived digitalis glycosides by the mammalian sodium and potassium transporting adenosine triphosphatase (Na,K-ATPase, or sodium pump), a plasma membrane enzyme with critical physiological importance in mammalian tissues, has raised the possibility that a mammalian analog of digitalis might exist. We previously isolated and structurally characterized from bovine hypothalamus a novel isomer of the plant glycoside, ouabain, which differs structurally only in the attachment site and/or the stereochemistry of the steroid moiety [Tymiak et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8189-8193]. Hamlyn and co-workers reported a molecule purified from human plasma which by mass spectrometry could not be distinguished from plant ouabain [Hamlyn et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6259-6263]. Since rhamnoside cardiotonic steroids are not known as natural products from mammalian sources, it became important to compare these two pure isolates to determine if the same or structurally distinct compounds has been found. Our results indicate that the human and bovine Na,K-ATPase-inhibitors are identical, but different from plant ouabain. This supports the notion that the human sodium pump may be under specific physiological regulation by a mammalian analog of the digitalis glycosides.

A rapid and versatile method for screening endothelin converting enzyme activity.

A flexible two-step protocol has been developed for high-throughput assays of endothelin converting enzyme (ECE) activity. The method is illustrated using endothelial cell monolayers and crude cell extracts as enzyme sources for conversion of exogenous substrates (big endothelin isopeptides) to their respective vasoactive products (endothelin isopeptides). Cleavage conditions have been optimized to minimize substrate utilization (reducing cost) while maintaining sensitivity and selectivity for mature endothelin detection in receptor- and antibody-based assays. Endothelins are quantified in a separate step and comparable estimates of mature endothelin formation are obtained using both EIA and radioreceptor assays in 96-well formats. Human umbilical vein (HUVEC) and human aorta (HAEC) endothelial cells characterized by this method are found to preferentially convert the big endothelin-1 (big ET-1) isopeptide through a membrane-bound, thiorphan-insensitive, and phosphoramidon-sensitive zinc metalloendopeptidase. Both intact cells and membrane preparations used as the enzyme source predict similar IC50 values for phosphoramidon inhibition of ECE (ca 1 microM). The procedure described is simple, rapid, and suitable for high-volume screening.

Physicochemical characterization of a ouabain isomer isolated from bovine hypothalamus.

Recent reports have shown the presence of a ouabain-like inhibitor of Na+/K(+)-ATPase in humans. We have purified a bovine hypothalamic Na+/K(+)-ATPase inhibitory factor (HIF) by using affinity chromatography combined with HPLC. This inhibitor has a molecular weight of 584 as determined by ion-spray mass spectrometry, making it isobaric with ouabain. Glycosidase treatment or acid hydrolysis of HIF released only L-rhamnose, the hexose isomer found in ouabain, as detected by chiral GC/MS. Additionally, enzymatically generated desrhamnosyl HIF was found to have a molecular weight of 438, as does ouabagenin, the aglycone of ouabain. HIF and its aglycone were indistinguishable from ouabain and ouabagenin, respectively, by reversed-phase HPLC retention times. However, derivatization with naphthoylimidazole followed by HPLC revealed different retention times for naphthoylation products of HIF and ouabain. Subsequent CD spectroscopy on isolated naphthoylation products of HIF and ouabain confirmed that they were different. This study provides chromatographic and spectroscopic evidence that ouabain and HIF are isomeric cardenolides. The structural difference is presumed to account for the significant differences in biological properties observed for HIF and ouabain.

Isolation and identification of lysophosphatidylcholines as endogenous modulators of thromboxane receptors.

Inhibition of thromboxane receptor radioligand binding to human platelet membranes has been employed as the basis for a radioreceptor assay designed to measure thromboxane receptor binding activity in samples of biological fluids. This method was used during phase 1 clinical evaluation of the thromboxane receptor antagonist SQ 30,741. Frequently, baseline plasma samples as well as plasma samples from placebo-treated subjects showed significant inhibition of radioligand binding in the radioreceptor assay, suggesting the presence of endogenous thromboxane receptor ligands. This receptor binding activity was stable and could be monitored in blood from normal volunteers using a modification of the radioreceptor assay. In order to identify the substance responsible for the observed activity, the activity present in pooled bovine blood was isolated and evaluated by a combination of FAB/MS, 1H-NMR, 13C-NMR and co-injection with reference standards on HPLC. Several endogenous thromboxane receptor ligands were identified as L-alpha-lysophosphatidylcholine (LPC) species. One major species, palmitoyl-LPC, contracted isolated rat aortic spirals, and these contractions could be delayed or prevented, but not reversed by the thromboxane receptor antagonist SQ 29,548. Palmitoyl-LPC slightly potentiated aortic contractions induced by the thromboxane receptor agonist, U-46,619, and diminished in a concentration-dependent manner the antagonism by SQ 29,548 of contractile responses to U-46,619. These findings are consistent with a potential for LPC species to bind and activate thromboxane receptors.

Dactylocyclines, novel tetracycline derivatives produced by a Dactylosporangium sp. I. Taxonomy, production, isolation and biological activity.

A screen for antibiotics with activity against tetracycline-resistant microorganisms has led to the isolation of Dactylosporangium sp. (ATCC 53693), a producer of several novel tetracycline derivatives. The major fermentation products, dactylocyclines A and B, were purified and MIC values determined against tetracycline-resistant and tetracycline-sensitive Gram-positive bacteria. The dactylocyclines represent the first naturally occurring tetracycline C2 amides which lack cross resistance with tetracycline.

Dactylocyclines, novel tetracycline derivatives produced by a Dactylosporangium sp. II. Structure elucidation.

Fermentation of Dactylosporangium sp. (ATCC 53693) produces a mixture of tetracycline derivatives from which several related tetracycline glycosides, the dactylocyclines, were isolated and their structures determined. The most abundant glycoside in initial fermentations was found to be dactylocycline A. Each glycoside proved to be acid sensitive and readily hydrolyzed to a common aglycone, dactylocyclinone. While the aglycone was cross resistant with tetracycline, the dactylocyclines proved active against certain tetracycline-resistant organisms.

Dactylocyclines, novel tetracycline derivatives produced by a Dactylosporangium sp. III. Absolute stereochemistry of the dactylocyclines.

The dactylocycline antibiotics were found through circular dichroism measurements, NMR spectroscopy and chemical transformations to possess the usual tetracycline family absolute configuration at carbons 4, 4a, 5a and 12a. The absolute stereochemistry about the C-6 carbon, however, was the reverse of that found with previously investigated tetracyclines.

Quantitation of lysophosphatidylcholine molecular species in rat cardiac tissue.

We have developed a rapid and sensitive procedure for isolation and measurement of 1-acyllysophosphatidylcholine (LPC) species in rat myocardial tissue. Tissues were spiked with heptadecanoyl-LPC internal standard and extracted with chloroform/methanol. The chloroform phase was dried, resuspended in chloroform/propan-2-ol (2/1, v/v), and applied to an aminopropyl-bonded phase (Bond Elut) column. Following stepwise elution with several solvent mixtures, the LPC fraction (ethyl acetate/methanol, 4/6, v/v) was separated by HPLC with direct quantitation of palmitoyl-LPC (P-LPC), oleoyl-LPC (O-LPC), and stearoyl-LPC (S-LPC), using an evaporative light scattering mass detector. Calibration curves were generated for each individual LPC species. Recoveries of added [14C]LPC and of heptadecanoyl-LPC internal standard after extraction and chromatography were 85.8 +/- 1.9% (mean +/- SE, N = 10) and 83.4 +/- 1.8% (N = 15), respectively. This assay showed satisfactory sensitivity, reproducibility, and accuracy for measurement of LPC species in rat myocardial tissue. The major molecular species of LPC in rat myocardium were found to be P-LPC and S-LPC, which were two- to sixfold as abundant as O-LPC. In isolated, crystalloid-perfused rat hearts the time of perfusion was found to significantly influence the content of P-LPC (0 min, 252 +/- 10; 15 min, 178 +/- 10, P less than 0.001, compared with 0 min; 40 min, 131 +/- 4, P less than 0.001; and 70 min, 129 +/- 4, P less than 0.001; nmol/g dry weight), but not the content of O-LPC and S-LPC. The method will be useful for studying the participation of LPC species in physiology, pathophysiology, and therapeutics.

Vascular A10 cell membranes contain an endothelin metabolizing neutral endopeptidase.

We have investigated the possible presence of endothelin-metabolizing neutral endopeptidase (NEP, EC 3.4.24.11) on A10 cell membranes using [125I]-ET-1 binding and direct measurements of NEP. NEP activity of A10 cell membranes has been compared to that of solubilized rat kidney brush border membranes (KNEP). Specific [125I]-ET-1 (50 pM) binding (defined with 100 nM ET-1) to A10 cell membranes was increased in a concentration dependent manner by the selective NEP inhibitors thiorphan, phosphoramidon, and SQ 28,603 [(+/-)-N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-beta-alanine] with EC50 values of 9.4, 28.4, and 5.7 nM respectively. At equilibrium (150 min), 70% more specific binding was apparent in the presence of these inhibitors. Phosphoramidon (2 microM) did not alter Bmax values, but it decreased the apparent KD for [125I] ET-1 from 63 (+/- 3) to 27 (+/- 2) pM. Thiorphan, phosphoramidon, and SQ 28,603 inhibited A10 cell NEP activity with IC50 values of 5.3, 36.5, and 6.0 nM respectively, which was similar to values obtained with KNEP (3.6, 22.6, and 3.5 nM). ET-1 inhibited A10 cell NEP, and KNEP with IC50 values of 30 and 21.3 microM respectively. The order of inhibitory potencies: ET-3 greater than ET-1 = ET-2 greater than or equal to sarafotoxin-6b was similar for both systems. These data suggest A10 cell membranes contain a NEP which has similar characteristics to NEP 24.11, and which actively metabolizes [125I]-ET-1.

Janthinocins A, B and C, novel peptide lactone antibiotics produced by Janthinobacterium lividum. II. Structure elucidation.

The structures of janthinocins A, B and C, three novel macrocyclic peptide lactone antibiotics isolated from fermentations of Janthinobacterium lividum, were determined. The janthinocins are of particular interest because they contain three amino acid residues that have not previously been reported in natural products: Each contains erythro-beta-hydroxy-D-leucine while janthinocins A and B also contain beta-hydroxytryptophan and beta-ketotryptophan, respectively.

Lysobactin, a novel antibacterial agent produced by Lysobacter sp. I. Taxonomy, isolation and partial characterization.

A new antibacterial agent, lysobactin, has been isolated from a species of Lysobacter (ATCC 53042). The antibiotic was recovered from the Lysobacter cell mass by extraction and reversed phase chromatography. Lysobactin is a dibasic peptide with marked activity against Gram-positive aerobic and anaerobic bacteria.