Failure of GPI compounds to display neurotrophic activity in vitro and in vivo.

The aim of this study was to evaluate the neurotrophic and neuroprotective properties of a series of immunophilin ligands and to assess the potential involvement of FK506 Binding Protein 12 kDa (FKBP12) rotamase inhibition in this activity. Both FK506 and rapamycin induced a potent inhibition of the FKBP12 rotamase activity (pIC(50) values of 7.3 and 7.4, respectively) but only a modest inhibition was observed with 1-(3,3-dimethyl-2-oxo-pentanoyl)-pyrrolidine-2-carboxylic acid S-3-pyridin-3-yl-propyl ester (GPI 1046) (5.8), its N-oxide (5.4) and thioester (6.3) analogues. Compared to nerve growth factor, all these immunophilin ligands only induced marginal increases in neurite outgrowth of rat dissociated newborn dorsal root ganglia cells. Furthermore, systemic administration of GPI 1046 and its N-oxide and thioester analogues failed to prevent striatal dopamine depletion induced by acute or chronic i.p. treatment with 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP). These results suggest that inhibition of FKBP12 rotamase activity is not predictive for neurotrophic and neuroprotective properties of immunophilin ligands and question their therapeutic utility in neurodegenerative diseases like Parkinson's disease.

Acute stress in rats produces a rapid and sustained increase in prostacyclin production in aortic tissue: dependence on corticosterone.

Immobilization stress in rats is accompanied by a fast and transient increase in plasma catecholamines (CA) and a slow but more sustained increase in plasma corticosterone (CORT) levels. CA are reported to increase prostacyclin (PGI2) secretion in endothelial cells in culture and in isolated vascular tissue. The present study was undertaken to determine a) whether short term immobilization stress in rats affects PGI2 synthesis in aortic tissue and b) whether such effects are dependent on the maintenance of an intact hypothalamic-pituitary-adrenal axis. Male Sprague Dawley rats were immobilized for 5, 15 and 30 min., killed by a blow to the head and aortic rings immediately prepared and incubated in oxygenated Krebs-Ringer buffer at 37 degrees C. Secretion of PGI2 was assessed by measuring the amount of 6-keto-PGF1 alpha, the stable hydrolysis product of PGI2, formed in the incubation medium during 90 min. Rats immobilized for 15 min demonstrated a six to seven times increase in plasma ACTH and a more than ten times increase in plasma CORT levels. Aortic rings from stressed rats also demonstrated a significant and sustained (2 hours) increase in PGI2 secretion when compared with tissue from unstressed rats (68 pg 6-keto-PGF1 alpha/mg tissue per min vs 35 pg 6-keto-PGF1 alpha/mg tissue per min during the first hour). Shorter (5 min) or longer (30 min) periods of stress gave comparable results. Adrenalectomy (ADX) carried out 7 days prior to immobilization, did not affect the baseline secretion of the eicosanoid, but completely prevented the increase in PGI2 by immobilization stress. Administration of 2 micrograms/kg and 200 micrograms/kg i.a. of CORT to ADX-rats produced plasma levels of the hormone (36 +/- 12 ng/ml and 934 +/- 366 ng/ml respectively) comparable to those seen in unstressed and stressed rats. However only the higher dose of CORT when administered immediately prior to stress, restored the effect of immobilization stress on PGI2 synthesis in aortic tissue of ADX-rats. Administration of the protein synthesis inhibitor cycloheximide (1 mg/kg) prior to stress did not affect the increase in PGI2 secretion. In contrast to restraint stress, co-administration of ACTH and NE which also raised plasma levels of ACTH, CORT and NE to peak stress levels, did not increase prostacyclin production in aortic tissue. In conclusion, our findings demonstrate that short periods of immobilization stress produce a rapid and sustained increase in PGI2 synthesis in the rat aorta, provided plasma levels of CORT are elevated.(ABSTRACT TRUNCATED AT 400 WORDS)

Formation and degradation of 1-(ethyl)-1-(2-hydroxyethyl) aziridinium chloride in aqueous media--a comparative NMR study.

The formation and decomposition of 1-(ethyl)-1-(2-hydroxyethyl) aziridinium chloride in aqueous media (10 mM solutions) was studied by NMR spectroscopy. Compared to 1-diethyl aziridinium chloride which lacks the hydroxyl group, the degradation of the title compound at 37 degrees C and pH = 7.4 is about 10 times faster. However, liquid nitrogen frozen solutions of both aziridinium salts are stable for long periods of time (1-6 months).

The use of agar gel as a basic reference material for calibrating relaxation times and imaging parameters.

The relaxation properties of agar gels render it a potentially useful basic reference material for calibrating the NMR equipment. The T1 and T2 values are close to the values observed for most biological tissues; they are stable and can be varied by controlling the concentration of MnCl2. The temperature, concentration, and volume dependence of T1 and T2 were studied.

In vitro metabolism of procyclidine in the rat.

Procyclidine, 1-cyclohexyl-1-phenyl-3-(1-pyrrolidinyl)-1-propanol, was incubated with the 9000g supernatant fraction of rat liver homogenates, fortified with a NADPH generating system. Three major metabolites were isolated from the incubation mixture. They were identified as 1-(cis-4-hydroxycyclohexyl)-1-phenyl-3-(1-pyrrolidinyl)-1-propanol, 1-(trans-4-hydroxycyclohexyl)-1-phenyl-3-(1-pyrrolidinyl)-1-propanol, and (1R*, 3R*, 7S(R?)*)-1-(trans-3-hydroxycyclohexyl)-1-phenyl-3-(1-pyrrolidinyl) -1-propanol. The latter has not been detected previously in rat urine and probably represents an intermediate metabolite.

Isolation and identification of eight procyclidine metabolites from rat urine.

After intraperitoneal administration of procyclidine, eight metabolites were isolated from rat urine. They were identified as 1-(4-oxocyclohexyl)-1-phenyl-3-(1-pyrrolidinyl)-1-propanol, 1-(cis-4-hydroxycyclohexyl)-1-phenyl-3-(1-pyrrolidinyl)-1-propanol, 1-(trans-4-hydrocyclohexyl)-1-phenyl-3-(1-pyrrolidinyl)-1-propanol , (1R,3R,4S,7R)- and (1R,3R,4S,7S)-1-(cis-3,cis-4-dihydroxycyclohexyl)-1-phenyl-3-(1-py rrolidinyl)- 1-propanol, (1R,3R,4R,7R)- and (1R,3R,4R,7S)-1-(cis-3,trans-4-dihydroxycyclohexyl)-1-phenyl- 3-(1-pyrrolidinyl)-1-propanol, and one of both (1R,3S,4R,7R)- or (1R,3S,4R,7S)- 1-(trans-3,trans-4-dihydroxycyclohexyl)-1-phenyl-3-(1-pyrrolidinyl )-1-propanol by comparative TLC, GLC-MS and 13C-NMR spectroscopy.

Experimental protocol for tissue discrimination in vitro by N.M.R.

We have examined the n.m.r. relaxation times T1 and T2 of water protons for liver (mouse, human) and brain (mouse) at different temperatures and subjected to various conditions of conservation and degeneration. After tissue degeneration, T1 and T2 behave differently and their variations are characteristic of each tissue type. The results show that the initial values at +4 degrees C are consistent when the experimental protocol formulated in this study is followed.

N-acetyl-(1-amino-2-naphthol-6-sulphonic acid), a common metabolite of sunset yellow FCF, orange GGN and 1-amino-2-naphthol-6-sulphonic acid in rat urine.

Sunset Yellow FCF, Orange GGN and 1-amino-2-naphthol-6-sulphonic acid (ANSA) were administered to male Wistar rats by stomach intubation. Aromatic sulphonic acids excreted in 24-h urines after enzymatic cleavage of the azo link in the dyes were isolated using a thoroughly elaborated ion-pair extraction method and further separated by means of a reversed-phase ion-pair liquid chromatographic system. Blank 24-h rat urines were extracted and run at the same time under identical analytical circumstances. In addition to the peaks corresponding to sulphanilic and metanilic acid and their N-acetylated derivatives, which are metabolites arising from Sunset Yellow FCF and Orange GGN, respectively, an important common peak appeared on the chromatograms, which was absent from the blank urine extracts. After analysis of 24-h urine of rats that had received ANSA, a peak with the same retention time as this unknown common peak could be detected under the same liquid chromatographic conditions, the retention time of which was different from that of the ANSA standard. However, after derivatization of the ANSA standard with acetic anhydride, followed by liquid chromatographic examination of the derivatised mixture, two peaks appeared on the chromatogram, the first of which had the same retention time and the same UV-spectrum as the unknown common peak in rat urine extracts. By means of semi-preparative liquid chromatographic separation and isolation, followed by further purification over a cation-exchange resin, the compound corresponding to the unknown common peak could be identified by FT-PMR spectroscopy as N-acetyl-ANSA.