Determination of N-acetylation phenotyping in a Greek population using caffeine as a metabolic probe.

Studies of isoniazid, the well known antituberculosis drug, have revealed that N-acetylation polymorphism, is of great clinical importance. In humans, N-acetylation is one of the most important pathways in the inactivation of isoniazid. Caffeine, which is also biotransformed by N-acetylation, has been widely used as an in vivo probe for the assessment of N-acetyltransferase polymorphism. The activity of N-acetyltransferase can be estimated from the urinary metabolic ratio of two caffeine metabolites, namely, 5-acetylamino-6-formylamino-3-methyluracil (AFMU), and 1-methylxanthine (1X) after the ingestion of caffeine. In the present study caffeine was used as a metabolic probe to determine N-acetyltransferase polymorphism in 83 healthy Greek volunteers by means of the molar ratio of AFMU and IX determined in urine following ingestion of 200 mg caffeine. Frequency distribution analysis of the metabolic ratios AFMU/1X revealed two distinct groups with 66.3% (n = 55) slow acetylators and 33.7 % (n = 28) rapid acetylators. No statistically significant difference was detected between slow and fast acetylators in terms of gender, smoking habits and caffeine-intake habits. These results are in agreement with previous studies on N-acetyltransferase activity in Caucasians using caffeine as a metabolic probe. They also agree with reports on N-acetyltransferase activity in Greek tuberculosis patients using isoniazid as a metabolic probe. Thus, the use of caffeine as a metabolic probe is a reliable method for the assessment of N-acetyltransferase activity in the Greek population.

Isolation and characterization of an endogenous peptide from rat brain interacting specifically with the serotonergic 1B receptor subtypes.

The existence of endogenous compounds interacting with the serotonergic system was previously postulated. In the present work, rat brain tissues were extracted by acidic and organic procedures. The resulting extract was tested for its capacity to interact with the binding of [3H]5-hydroxytryptamine ([3H]5-HT) to 5-HT1 receptors. Compounds responsible for the observed inhibitory activities were isolated and purified by high pressure liquid chromatography. A tetrapeptide corresponding to a novel amino acid sequence Leu-Ser-Ala-Leu (LSAL) was identified. It reduces the binding of [3H]5-HT to 5-HT1 receptors at low concentration (IC50 = 10(-10) M). This effect corresponds to a specific interaction at 5-HT1B receptors since LSAL does not significantly affect other neurotransmitter bindings. LSAL appears heterogeneously distributed throughout the brain (hippocampus > cerebellum > striatum > brain stem) and in peripheral tissues (kidney > lung > stomach > blood > liver > spleen). Two other peptides, Leu-Ser (LS) and Ala-Leu (AL), were also purified. They hardly affected [3H]5-HT binding compared with LSAL. They presumably represent degradation products of the functional peptide LSAL. The fact that LSAL interacts specifically with 5-HT1B receptors that inhibit the release of neurotransmitters and particularly that of 5-HT itself suggests that this peptide may be involved in mechanisms controlling 5-HT neurotransmission and, accordingly, may play an important role in pathophysiological functions related to 5-HT activity.

A new peptide, 5-HT-moduline, isolated and purified from mammalian brain specifically interacts with 5-HT1B/1D receptors.

5-HT-Moduline (Leu-Ser-Ala-Leu) is a new endogenous peptide purified from rat brain which interacts specifically with 5-HT1B/1D receptors. The binding interaction of 5-HT-Moduline with 5-HT1B/1D receptors appeared to be a non-competitive process, since the Bmax value of [125I] cyanopindolol binding on rat brain cortical membranes was decreased without modification of the Kd. This interaction was conserved on NIH 3T3 cells expressing the 5-HT1B receptor (IC50 = 10(-11)M) suggesting that the binding site for 5-HT-Moduline is localized on the 5-HT1B receptor protein. The observed interaction may lead to functional alterations of 5-HT1B/1D receptors known to play an important role in regulating the release of 5-HT from serotonergic nerve terminals (autoreceptors) as well as the release of other neurotransmitters (heteroreceptors).

Postnatal development of 5-HT1 receptors: [3H]5-HT binding sites and 5-HT induced adenylate cyclase activations in rat brain cortex.

The postnatal development of the 5-HT1 receptor system was studied in young rat brain cortex from birth to adulthood (14 successive ages). The high-affinity binding of [3H]5-HT was low at birth but developed markedly between the 8th and the 15th day postnatally. The basal adenylate cyclase activity produced 50 pmoles cAMP/mg protein/min at birth and increased from the 8th to the 15th day. 5-HT could stimulate the adenylate cyclase activity in adult rat brain cortex with two different affinity constants: Km = 1 nM and Km = 0.5 microM; these low- and high-affinity constants presumably correspond to 5-HT1A and 5-HT1non-A.non-B.non-C (5-HT1D) respectively. These two activities developed parallelly from the 14-15th to the 28th day. The 8-hydroxy-2-(di-n-propylamino-tetralin) (8-OH-DPAT)-induced activity described a curve similar to the one that corresponded to 10 microM 5-HT. These results establish that 5-HT1A and 5-HT1non-A.non-B.non-C receptors mainly develop during the synaptogenesis.