Inhibition of endothelial cell prostaglandin H synthase gene expression by naproxen.

Naproxen is a potent anti-inflammatory drug whose action is attributed to inhibition of prostaglandin biosynthesis. In view of our recent discovery that aspirin and sodium salicylate are capable of reducing cellular levels of prostaglandin H (PGH) synthase mRNA, we have evaluated the effect of naproxen on PGH synthase protein and mRNA levels in cultured human umbilical vein endothelial cells (HUVEC). PGH synthase mRNA levels were quantified by a competitive polymerase chain reaction (PCR) assay; protein was assessed by Western blotting. Naproxen decreased the PGH synthase protein level in HUVEC in a concentration-dependent manner. It abolished entirely the 70 kDa PGH synthase subunit at 5 micrograms/ml. It appears more effective in blocking interleukin-1 inducible PGH synthase levels. Naproxen also inhibited the synthase mRNA level in a concentration-dependent manner; levels were reduced by 33% at 5 micrograms/ml and 60% at 30 micrograms/ml naproxen. These results indicate that naproxen, like the salicylates, inhibits PGH synthase levels in cultured endothelial cells either by inhibiting transcription of the PGH synthase gene or by destabilizing its messenger RNA.

In vivo metabolism of the cardiovascular toxin, allylamine.

Previous evidence from this laboratory demonstrated that allylamine, a known cardiovascular toxin, is metabolized in vitro to acrolein, which has been hypothesized to act as a distal toxin. In this study, 3-hydroxypropylmercapturic acid was isolated and identified by MS, NMR, and 2D-NMR spectroscopy as the sole urinary metabolite of allylamine metabolism in vivo. Parallel experiments showed reduced glutathione (GSH) depletion in several organs (most marked in aorta, blood, and lung), which is consistent with GSH conjugation of the proposed acrolein intermediate. These findings indicate that allylamine was metabolized in vivo to a highly reactive aldehyde which was converted to a mercapturic acid through a GSH conjugation pathway; the exact mechanisms of cellular damage remain unclear.

Isolation and structure of a cytotoxic epoxy sterol from the marine mollusc Planaxis sulcatus.

A novel epoxy sterol isolated from the marine mollusc Planaxis sulcatus has been identified as 9 alpha,11 alpha-epoxycholest-7-ene-3 beta,5 alpha,6 beta-triol by spectrometric methods.

Isolation, synthesis, and evaluation of a series of indencarbazates as hypotensive agents.

Two indencarbazates, 1 and 2, were isolated from the sponge Cliona caribboea. These compounds were found to possess mild hypotensive activity. A series of analogues of 1 was synthesized in order to study the structure-activity relationship of this unique class of compounds. A variety of structural changes did not result in a consistent pattern of biological activity.

3-Hydroxypropylmercapturic acid: a biologic marker of exposure to allylic and related compounds.

3-Hydroxypropylmercapturic acid [3-OHPrMCA, S-(3-hydroxypropyl)-N-acetyl-L-cysteine] was quantitatively measured by high-performance liquid chromatography (HPLC) in the urine of rats given allylamine.HCl (5, 25, 50, 100 and 150 mg kg-1), acrolein (13 mg kg-1), allylalcohol (64 mg kg-1), allylchloride (76 mg kg-1), allylbromide (120 mg kg-1), allylcyanide (115 mg) and cyclophosphamide (160 mg kg-1) by gavage in water. 3-OHPrMCA was measured by HPLC in 24-h urine collections; the lower detection limit was 1.25 micrograms or 5.6 nmol ml-1. Various doses of allylamine resulted in 3-OHPrMCA excretion at a fairly constant percentage of the dose, ca. 44-48% at 0-24 h and 3% at 24-48 h, indicating rapid metabolism through glutathione conjugation in the first 24h. Similarly, 3-OHPrMCA was recovered in the urine of rats given acrolein (78.5%), allylalcohol (28.3%), allylchloride (21.5%), allylbromide (3.0%), allylcyanide (3.7%) and cyclophosphamide (2.6%). These data indicate that 3-OHPrMCA can be used as a marker of exposure to allylic and other compounds that lead to the metabolic formation of acrolein.

A role for a new vascular enzyme in the metabolism of xenobiotic amines.

Although it has long been thought that environmental toxins may play an underlying role in vascular diseases such as atherosclerosis, this concept is not supported by any clear-cut experimental evidence of toxic metabolism by cardiovascular enzymes. In this study, we demonstrate that allylamine, a selective cardiovascular toxin in vivo, is actively metabolized in vitro by a purified vascular enzyme (semicarbazide-sensitive amine oxidase), which has been localized recently to vascular smooth muscle cells. Oxidative deamination of allylamine to a highly toxic aldehyde, acrolein, was blocked through enzyme inhibition by semicarbazide-sensitive amine oxidase suggests that this vascular enzyme's physiological role may include metabolism of exogenous amines.

Simultaneous determination of myocardial creatine phosphate and adenine nucleotides by reversed-phase HPLC.

An isocratic HPLC system has been developed which allows for the rapid (single run of 20 min) measurement of creatine phosphate (PCr) and adenine nucleotides (ATP, ADP and AMP) in extracts from freeze-clamped and freeze-dried myocardial tissues. The separation was achieved at room temperature by using a RP18 column and a dual variable wavelength spectrophotometer, set at 210 and 254 nm. The solvent was 30 mM potassium dihydrogen phosphate, 15 mM tetrabutylammonium hydrogen sulfate, pH 6.7, 19% (v/v) acetonitrile. A distinct separation (confirmed with the retention time of standard sample) of these high energy compounds was achieved. Standard curves were linear. In isolated rat hearts the following values were obtained (mumol/g dry wt, mean +/- SEM): ATP 21.5 +/- 1.3, ADP 4.6 +/- 0.2, AMP 1.5 +/- 1.1 and PCr 32.5 +/- 1.3; which are consistent with previously published values for high energy compounds in this tissue.

Aspirin inhibits interleukin 1-induced prostaglandin H synthase expression in cultured endothelial cells.

Prostaglandin H (PGH) synthase (EC 1.14.99.1) is a key enzyme in the biosynthesis of prostaglandins, thromboxane, and prostacyclin. In cultured human umbilical vein endothelial cells, interleukin 1 (IL-1) is known to induce the synthesis of this enzyme, thereby raising the level of PGH synthase protein severalfold over the basal level. Pretreatment with aspirin at low concentrations (0.1-1 micrograms/ml) inhibited more than 60% of the enzyme mass and also the cyclooxygenase activity in IL-1-induced cells with only minimal effects on the basal level of the synthase enzyme in cells without IL-1. Sodium salicylate exhibited a similar inhibitory action whereas indomethacin had no apparent effect. Similarly low levels of aspirin inhibited the increased L-[35S]methionine incorporation into PGH synthase that was induced by IL-1 and also suppressed expression of the 2.7-kilobase PGH synthase mRNA. These results suggest that in cultured endothelial cells a potent inhibition of eicosanoid biosynthetic capacity can be effected by aspirin or salicylate at the level of PGH synthase gene expression. The aspirin effect may well be due to degradation of salicylate.

Structure of 3-O-acetylgibberellin A3.

C21H24O7, Mr = 388.4, orthorhombic, P2(1)2(1)2(1), a = 13.279 (10), b = 15.615 (10), c = 9.427 (8) A, V = 1954.7 A3, Z = 4, D chi = 1.319 g cm-3, lambda(Mo K alpha) = 0.71069 A, mu = 1.0 cm-1, F(000) = 824, T = 293 K, R = 0.049 for 1805 observed reflections. The gibberellane skeleton shows the conformational variety characteristic of many other gibberellin derivatives. Ring A is in a sofa conformation while the five-membered ring B assumes an intermediate conformation between an envelope and a half-chair. The lactone ring is in an ideal envelope conformation and shows a marked difference in its two C-O distances. Each molecule is hydrogen bonded to two others to form continuous spiral chains along the c axis.

A pyrazoline derivative of eunicin acetate.

The present crystal structure determination established spiro [¿3a,4,5,6,7,8,11,12,13,14,15,15a-dodecahydro-6,10,14-trimet hyl-2-oxo-5, 15-epoxy-3H-cyclotetradeca-[b]furan¿-3,3'-1'-pyrazoline]-6-yl acetate, C23H34N2O5, as a pyrazoline derivative of eunicin acetate. The spiro substitution of the pyrazoline ring causes elongation of the bonds within the lactone ring and also shortening of the carbonyl bond. The cembranolide skeleton is slightly more bent than that observed in the parent eunicin molecule.

Isolation of the cembranolide diterpenes dihydrosinularin and 11-epi-sinulariolide from the marine mollusk Planaxis sulcatus.

Marine mollusks have been identified as a rich source of novel compounds. The diversity of the chemical nature of these compounds can be partially attributed to the dietary sources inasmuch as mollusks are known to concentrate selectively compounds present in their diet. Although cembranolide diterpenses have been isolated in ever-growing numbers from a variety of marine as well as terrestrial sources, there is only one report about their presence in a marine mollusk. In this communication, we report the isolation of dihydrosinularin and 11-epi-sinulariolide from the mollusk Planaxis sulcatus Born. Dihydrosinularin and 11-epi-sinulariolide acetate have been isolated previously from soft corals which, together with gorgonians, are the richest sources of marine cembranoids. It should be pointed out that this is the first report on the occurrence of the parent 11-epi-sinulariolide in a marine invertebrate. It is expected these diterpenes are of dietary origin although the possibility of biotransformation of a precusor by the mollusk cannot be ruled out at this time. Both dihydrosinularin and 11-epi-sinulariolide have been reported to have marginal antineoplastic activity against P-388 lymphocytic leukemia.

Differentiation-associated expression of prostaglandin H and thromboxane A synthases in monocytoid leukemia cell lines.

To elucidate the differentiation-associated expression of enzymes catalyzing arachidonic acid metabolism, we measured arachidonate metabolites by reverse-phase high pressure liquid chromatography in monocytoid leukemia (ML-1, THP-1, and U937) and myeloid leukemia (KG-1) cell lines. Undifferentiated ML-1 or THP-1 cells produced trace amounts of eicosanoids via the cyclooxygenase (COX) and lipoxygenase (LOX) pathways. Upon differentiation induced by phorbol ester (phorbol 12-myristate 13-acetate [PMA]), metabolites via the COX pathway were increased by 100-fold in ML-1 and THP-1 cells, while the LOX products remained barely detectable. All the COX metabolites were elevated, but thromboxane A2 (TXA2) formation was threefold higher in ML-1 cells than in THP-1 cells. Similar time-related increases in COX metabolites were observed in THP-1 cells induced to differentiate with retinoic acid. Undifferentiated U937 cells were capable of generating a much higher quantity of COX products than ML-1 or THP-1 cells, but, upon PMA-induced differentiation, COX products were increased by only two-fold to threefold over the undifferentiated cells and the total COX products in differentiated U937 cells were only one-seventh of those produced by differentiated ML-1 or THP-1 cells. KG-1 cells had an entirely different metabolic profile. They produced a large quantity of a metabolite coeluted with prostaglandin D2, and PMA had no effect on inducing changes in arachidonic acid (AA) metabolism. Increased COX metabolite formation in differentiated THP-1 and ML-1 cells was due to an enhanced level of prostaglandin H synthase enzyme mass, as measured by Western blot analysis. The TXA synthase activity was also increased by approximately 100-fold in PMA-induced ML-1 cells and 10-fold in THP-1 cells. These findings indicate that increased expression of prostaglandin H and TXA synthase enzymes is a feature of differentiated monocytoid leukemia cell lines.