The triaminopyridine flupirtine prevents cell death in rat cortical cells induced by N-methyl-D-aspartate and gp120 of HIV-1.

Flupirtine, a triaminopyridine derivative, is a non-opiate centrally acting analgesic agent with muscle relaxant properties. Now we show that this drug displays a potent cytoprotective effect on neurons (rat cortical cells) treated with (i) the excitatory amino acid N-methyl-D-aspartate (NMDA) or (ii) with the human immunodeficiency virus type 1 (HIV-1) coat protein gp120. In the absence of the drug the two agents cause a > 90% reduction of cell viability after a 18 h incubation. During this period the DNA in the cells undergoes fragmentation and shows a pattern which is typical for cell death. If the neurons were preincubated with flupirtine for 2 h and subsequently exposed to the cytotoxic agents an almost complete protection was achieved. The cytoprotective effect of flupirtine in vitro was significant (above 10 microM). Because flupirtine displays almost no clinical side effects and in light of the data presented here, flupirtine may be a promising drug also for the treatment of NMDA-mediated neurodegenerative disorders in general and for the treatment of AIDS-related encephalopathy in particular.

A non-radioactive, sensitive method for the detection of DNA fragmentation in apoptotic cells (rat pheochromocytoma PC12 and rat cortical cells).

A new method is described which is suitable for reliably analysing apoptotic fragmentation in small amounts of DNA. After isolation, DNA was labelled with biotin-4-dUTP using Klenow polymerase. Then DNA was size-separated by agarose gel electrophoresis, blot transferred and subsequently visualized by the streptavidin alkaline phosphatase-BCIP/NBT procedure. This non-radioactive method was used to detect apoptotic DNA in rat pheochromocytoma PC12 cells, treated with tributyltin (1 nM). While only 30 ng of DNA is required for analysis of apoptotic DNA using the new blot technique, 100-fold more material is needed to identify the fragmentation of DNA after separation by agarose gel electrophoresis and direct staining with ethidium bromide. In a further set of experiments, rat cortical cells were incubated with human immunodeficiency virus type 1 viral glycoprotein of M(r) of 120 kDa (gp120) to induce apoptosis. More than 0.3 ng of gp 120/ml are required to detect apoptotic DNA by the direct procedure; only 0.1 ng gp120/ml or less were sufficient to document clear DNA fragmentation using the non-radioactive blotting technique described here. These results demonstrate that the new procedure can be used to analyse very small amounts of apoptotic DNA and shows that gp120-induced apoptosis can be measured at low concentrations of the viral protein.

A simple and reliable method for monitoring platelet serotonin levels in rats.

A simple and reliable method for individual monitoring of platelet serotonin in rats is developed. Platelet-rich plasma is prepared under standardized conditions from 1 mL of venous blood and the platelets are quantitatively separated by a highly reproducible procedure. Platelet serotonin content is determined spectrophotofluorometrically and the results are comparatively expressed per standardized platelet rich plasma sample (1.01 +/- 0.18 microgram), per mg of platelet protein (1.57 +/- 0.15 microgram) and per 10(9) platelets (2.16 +/- 0.38 micrograms). Normal distribution of platelet serotonin levels in a sample of 338 animals is shown. By use of the described method, the intraindividual stability of platelet serotonin concentration in rats is demonstrated for the first time.

The effects of immunomodulating peptidoglycan monomer and muramyl dipeptide on hepatic microsomal UDP-glucuronyltransferase and beta-glucuronidase.

The effects of immunomodulating peptidoglycans, peptidoglycan monomer (PGM) and muramyl dipeptide (MDP), on hepatic microsomal UDP-glucuronyltransferase (uridine diphosphoglucuronate glucuronosyl transferase, EC 2.4.1.17) and beta-glucuronidase (beta-D-glucuronide glucuronohydrolase, EC 3.2.1.31) were tested in female C57Bl mice. 4-Methylumbelliferone and p-nitrophenol were used as representative substrates for one functional form of UDP-glucuronyltransferase (GT1) and testosterone for the second functional form (GT2) of the enzyme. Both PGM and MDP were found to transiently inhibit the activity of UDP-glucuronyltransferase. There was no significant difference in the magnitude of inhibition of the two functionally different enzyme forms. The activity of microsomal beta-glucuronidase was tested using 4-methylumbelliferyl glucuronide and p-nitrophenyl glucuronide as substrates. Time dependent transient inhibition of beta-glucuronidase activity was observed with both peptidoglycans. In addition, the effect of MDP on cytochrome P-450 was tested, since we have shown previously that PGM affected this system. MDP decreased the content of cytochrome P-450 and inhibited the activity of related enzymes.

Metabolism of tryptophol in higher and lower plants.

Bacteria, thallophytes, and seed plants (107 species), supplied with exogenous indole-3-ethanol (tryptophol), formed one or more of the following metabolites: O-acetyl tryptophol, an unknown tryptophol ester (or a set of structurally closely related esters), tryptophol glucoside, tryptophol galactoside, indole-3-acetic acid (IAA), and indole-3-carboxylic acid. The unknown ester was formed by all species examined; O-acetyl tryptophol appeared sporadically in representatives of most major taxonomic groups. Tryptophol galactoside was found in the algae Chlorella, Euglena, and Ochromonas. The glucoside was formed by many eucaryotic plants, but not by bacteria; it was a significant tryptophol metabolite in vascular plants. IAA, if detectable at all, was usually a minor metabolite, as should be expected, if tryptophol oxidase responds to feedback inhibition by IAA. Indole-3-carboxylic acid, formed by a few fungi and mosses, was the only tryptophol metabolite detected which is likely to be formed via IAA.

Formation of Tryptophol Galactoside and an Unknown Tryptophol Ester in Euglena gracilis.

The unicellular alga Euglena gracilis Klebs ;Z' converted exogenous indole-3-ethanol (trytophol) to two major metabolites: tryptophol galactoside and an unknown compound, and to minor amounts of indole-3-acetic acid, tryptophol acetate, and tryptophol glucoside. The unknown was hydrolyzed to tryptophol by methanolic ammonia and should therefore be a tryptophol ester. The galactoside was identified as 2-(indol-3-yl)ethyl-beta-d-galactopyranoside. This structure was established by comparison with an authentic standard involving chromatographic methods, ultraviolet and mass spectroscopy, enzymic and acid hydrolysis, and identification of the galactose in the hydrolysate. By forming tryptophol galactoside, Euglena differs from the higher plants examined so far, for which the corresponding glucoside is the only sugar conjugate of tryptophol detected.

Metabolism of Tryptophan, Indole-3-acetic Acid, and Related Compounds in Parasitic Plants from the Genus Orobanche.

Metabolic reactions involving the aliphatic side chain of tryptophan were studied in the holoparasitic dicotyledonous plants Orobanche gracilis Sm., O. lutea Baumg., and O. ramosa L. Unlike known autotrophic plants, the parasite metabolized l-tryptophan directly to indole-3-carboxaldehyde, which was further converted to indole-3-methanol and indole-3-carboxylic acid. Independently, these metabolites were also formed from d-tryptophan, tryptamine, indole-3-lactic acid, and indole-3-acetic acid. As in autotrophic plants, tryptophan and tryptamine were also converted, via indole-3-acetaldehyde, to indole-3-acetic acid, indole-3-ethanol, and its glucoside. The branch of tryptophan metabolism relevant to auxin biogenesis and catabolism is, therefore, not rudimentary in Orobanche but even more complex than in autotrophic higher plants.

Preliminary investigations of the metabolism and pharmacological activity of beta-hydroxytryptamines in mammals.

beta-Hydroxytryptamine and beta-hydroxy-5-hydroxytryptamine were incubated with rat liver slices and oxidative deamination was established as the main route of metabolism: in both instances the corresponding indole-3-glycollic acids and indole-3-ethane diols were the major metabolites. However, the rates of deamination of beta-hydroxylated tryptamines, as measured manometrically, were found to be much slower than those of tryptamines nonhydroxylated in the side chain. The pharmacological activities of beta-hydroxylated tryptamines were tested in guinea-pigs on resistance of respiratory pathways, spontaneous respiration, electrocardiogram, blood pressure and isolated ileum, using tryptamine and 5-HT as reference substances. The effects of tryptamines hydroxylated in the side chain were in general similar to those of corresponding tryptamines but of much lower intensities; only in increasing the blood pressure was beta-hydroxytryptamine as active as tryptamine. The different reactions of these two groups of substances in the presence of some antagonists indicate that the receptors are probably not the same.

Fluctuation of 5-hydroxy-indole compounds in the urine of migrainous patients.

In 14 migrainous patients during different clinical phases the urinary excretion of 5-hydroxy-tryptamine (5-HT) and its main metabolite, 5-hydroxyindoleacetic acid (5-HIAA) were followed. In the course of headache attack a significant increase in 5-HIAA excretion rate was found; at the same time the excretion of 5-HT was not significantly changed. After migraine attack a very pronounced lowering in excretion rate of both 5-hydroxy-indoles occured. The reserpine provocation test was followed by an intensive enhancement of urinary 5-hydroxyindole excretion, but the decrease thereafter was less expressed than after spontaneous attack. These findings lend support to the theory of an abrupt fall in total plasma 5-HT as a trigger mechanism for the painful phase of migraine.

The formation of tryptophol glucoside in the tryptamine metabolism of pea seedlings.

Tryptamine was converted by etiolated pea seedlings into IAA, tryptophol, and an appreciable amount of an unknown metabolite. This latter compound was characterised by TLC and electrophoresis and identified, by mass spectrometry and enzymatic cleavage, as tryptophol glycoside: indole-3-ethyl-ß-D-glycopyranoside.

Indole-3-methanol-a metabolite of indole-3-acetic acid in pea seedlings.

Degradation of indole-3-acetic acid was investigated in etiolated pea shoots; the study was limited to indolic metabolites. The products formed were fractionated by column chromatography and identified by thin-layer chromatography and chemical methods. The pathway of indole-3-acetic acid degradation involving indole-3-aldehyde was found to be more significant than stated in literature, and indole-3-methanol was established as the major indolic metabolite.