Characterization and subcellular localization of L-[3H] carnitine binding sites in rat brain.

In the present study, we investigated the existence of a binding site for L-carnitine in the rat brain. In crude synaptic membranes, L-[3H]carnitine bound with relatively high affinity (KD = 281 nM) and in a saturable manner to a finite number (apparent Bmax value = 7.3 pmol/mg of protein) of binding sites. Binding was reversible and dependent on protein concentration, pH, ionic strength, and temperature. Kinetic studies revealed a Koff of 0.018 min-1 and a Kon of 0.187 x 10(-3) min-1 nM-1. Binding was highest in spinal cord, followed by medulla oblongata-pons > or = corpus striatum > or = cerebellum = cerebral cortex = hippocampus = hypothalamus = olfactory bulb. L-[3H]Carnitine binding was stereoselective for the L-isomers of carnitine, propionylcarnitine, and acetylcarnitine. The most potent inhibitor of L-[3H]carnitine binding was L-carnitine followed by propionyl-L-carnitine. Acetyl-L-carnitine and isobutyryl-L-carnitine showed an affinity approximately 500-fold lower than that obtained for L-carnitine. The precursor gamma-butyrobetaine had negligible activity at 0.1 mM. L-Carnitine binding to rat crude synaptic membrane preparation was not inhibited by neurotransmitters (GABA, glycine, glutamate, aspartate, acetycholine, dopamine, norepinephrine, epinephrine, 5-hydroxytryptamine, histamine) at a final concentration of 0.1 mM. In addition, the binding of these neuroactive compounds to their receptors was not influenced by the presence of 0.1 mM L-carnitine.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C activation and anti-amnesic effect of acetyl-L-carnitine: in vitro and in vivo studies.

Drugs belonging to different chemical classes having the ability to improve behavioral performance in animal learning and memory tests may share the common ability to stimulate protein kinase C activity in rat brain cortex. In vitro acetyl-L-carnitine (100 nM) promoted in rat brain cortex slices a significant increase in particulate activity associated with lower soluble protein kinase C activity and produced a direct stimulation of the enzyme in both the cortex and hippocampus. In vivo a significant increase in particulate protein kinase C activity was observed in the group of rats treated with 60 mg/kg acetyl-L-carnitine, a dose shown to be effective in improving the cognitive deficits induced by scopolamine in the Morris maze test. The results suggest that acetyl-L-carnitine increases particulate protein kinase C activity in the cortex both in vitro and in vivo. This effect in the in vivo experiments seems to be observed only with doses that are effective in improving the performance of rats in a spatial learning task.

Beneficial effects of a novel platelet-activating factor receptor antagonist, ST 899, on endotoxin-induced shock in mice.

The effect of ST 899, a novel platelet-activating factor (PAF) receptor antagonist, on serum tumor necrosis factor (TNF), interleukin-6 (IL-6), and interferon-gamma (IFN-gamma) production as well as on lethality in an experimental endotoxin shock model was investigated in C57BL/6 mice. In this model, animals receiving 40 mg/kg lipopolysaccharide (LPS) (Escherichia coli 055:B5) intraperitoneally were pretreated with ST 899 administered according to two different schedules. ST 899 pretreatment dose dependently reduced the mortality induced by LPS injection. The PAF receptor antagonist was also able to reduce significantly the LPS-induced increase in serum TNF. Although serum IL-6 levels were not affected, we found that ST 899, when administered intraperitoneally 60 min and intravenously 10 min prior to LPS challenge, had a tendency (at higher doses) to decrease circulating IFN-gamma levels. It is suggested that ST 899 may be beneficial, in combination with current therapies, in the treatment of diseases that involve overproduction of PAF, TNF, and IFN-gamma such as septic shock.

GABA-activated chloride currents of postnatal mouse retinal ganglion cells are blocked by acetylcholine and acetylcarnitine: how specific are ion channels in immature neurons?

The goal of this study was to clarify pharmacological properties of GABAA receptors in cells of the mouse retinal ganglion cell layer in situ. Spontaneous synaptic currents and responses to exogenous GABA were recorded from individual neurons in retinal whole mounts (postnatal days 1-3) or retinal stripe preparations (postnatal days 4-6). Drugs were applied by a fast local superfusion system. Current responses were measured with the patch-clamp technique in the whole-cell configuration. All cells responded to exogenous GABA (average EC50 and Hill coefficient: 16.7 microM and 0.95 respectively) and generated GABAergic synaptic currents in response to elevated KCl. GABA-induced currents of retinal ganglion cells were blocked by bicuculline, picrotoxin and Zn2+, as well as strychnine, and increased by pentobarbital, clonazepam and 3 alpha-hydroxy-5 alpha-pregnan-20-one. In some retinal ganglion cells GABA caused an increase in the frequency of spontaneous synaptic currents, which points to a partially depolarizing action of this traditionally inhibitory neurotransmitter in the neural retina. Our major observation is that acetylcholine and acetylcarnitine blocked or reduced GABAergic inhibitory postsynaptic currents and responses to exogenous GABA. This effect was seen in only a fraction of retinal ganglion cells and occurred in both the undesensitized and the desensitized state of the GABAA receptor. The block was voltage-independent and persisted during coapplication with the nicotinic and muscarinic acetylcholine receptor antagonists D-tubocurarine and atropine. In contrast to GABA-activated Cl- currents, glycine-activated Cl- currents remained unaffected by acetylcholine and acetylcarnitine.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated urinary excretion of endothelins in streptozotocin diabetic rats.

Endothelin-1,2 urinary excretion, has been determined in control and streptozotocin diabetic rats at different times after diabetes induction. Diabetic rats showed increased urinary excretion of endothelins as compared to control rats, already three days after diabetes induction and up to 20 weeks.

Decreased density of endothelin binding sites in adrenal glands but not in aorta, of long term infarcted rats.

Endothelins (Et-s) are biologically active peptides which play a physiological and pathological role in the cardiovascular regulation. The aim of our study was to verify, in a model of experimental long term myocardial ischemia (15 weeks) in rats, whether there was a modification in the ET binding sites of aorta and adrenal glands. Additionally, Ang II binding sites in adrenal glands were studied. The principal finding of the present study was the down-regulation of ET binding sites in adrenal glands of chronic infarcted rats, whereas no modification of binding parameters for Et-1, in thoracic aorta, nor for Ang II, in adrenal glands, were found.

Acetyl-L-carnitine effect on nerve conduction velocity in streptozotocin-diabetic rats.

Measurement of nerve conduction velocity (NCV) is a useful and sensitive tool for evaluating diabetes related neurological dysfunctions. The method used allows to monitor the parameter at different times in the same group of rats, so that it is possible to observe simultaneously the development of the damage in time, and to evaluate the improvement related to the treatment. The repeated oral treatment with acetyl-L-carnitine (ALC, CAS 5080-50-2) 250 mg/kg caused an improvement in NCV of the diabetic rats; the effect was higher when the treatment started early with respect to the diabetes induction. The improvement in NCV was constant in time and comparable from 2 to 6 weeks of the treatment. In conclusion, oral treatment with ALC was able to normalize the impairment of NCV in streptozotocin rats, the effect being constant in time from 2 to 6 weeks of treatment and up to 8 weeks after induction when administration started in early stage of diabetes (2-3 weeks after induction); however, at this time the NCV is already significantly decreased.

LPS-induced serum TNF production and lethality in mice: effect of L-carnitine and some acyl-derivatives.

The effect of L-carnitine and some of its acyl derivatives on serum TNF production and lethality in a murine experimental endotoxin shock model was investigated. In some instances, serum IL-6 production was also evaluated. In this experimental model, C57BL/6 mice received 30 mg/kg LPS (E. cell 055:B5) injected intraperitoneally, while L-carnitine and its derivatives were administered according to different schedules. Serum levels of TNF and IL-6 were evaluated 1 h following LPS injection. The treated animals were also monitored daily for differences in body temperature, feeding, and survival for 10 days after LPS injection. Although some derivatives were able to significantly affect TNF production, the marked decrease in serum TNF levels of LPS-treated mice was not paralleled by a substantial increase in survival.

Determination of pentoxifylline and its metabolites in human plasma by high-performance liquid chromatography with solid-phase extraction.

A simple and reliable method for the determination of pentoxifylline and its main metabolites in human plasma has been developed using high-performance liquid chromatography. After selective solid-phase extraction, pentoxifylline, its metabolites and an internal standard, 7-(2'-chloroethyl)theophylline, were separated on a 5-micron LiChrospher 100 RP-18 column using water-dioxan-acetonitrile (87:6.5:6.5, v/v/v) acidified with acetic acid (0.5%, v/v) as the mobile phase. The analytes were detected at 275 nm. The lowest detectable concentration for all analytes was 25 ng/ml; the recovery was 85%. The assay has been successfully applied to analysis of these compounds in human plasma after administration of an oral dose of 400 mg of pentoxifylline to healthy volunteers.

L-carnitine deficiency in AIDS patients.

OBJECTIVE: To evaluate carnitine (3-hydroxy-4-N-trimethyl-ammoniobutanoate) deficiency in AIDS patients by measuring serum total, free and short-chain carnitine concentrations. DESIGN: We conducted an open study. SETTING: All patients were seen at the Infectious Diseases Clinic, Università 'La Sapienza', Rome, Italy. PATIENTS, PARTICIPANTS: Twenty-nine AIDS patients, aged 27-41 years, with a previous history of drug use; and 14 healthy age- and sex-matched controls were studied. INTERVENTIONS: Study subjects were administered 500-800 mg zidovudine daily for 2 to 28 months (8 +/- 6 months). MAIN OUTCOME MEASURES: Carnitine deficiency was suspected in study participants prior to data collection because of previously reported cardiac symptoms, muscle weakness, hypometabolism and/or cachexia. RESULTS: A marked decrease in total and free carnitine was observed in 21 (72%) subjects. Nine of these patients also had low levels of short-chain carnitine. CONCLUSIONS: AIDS patients may become carnitine-depleted and therefore at risk for alterations in fatty-acid oxidation and energy supply.

Rapid determination of amino acids by high-performance liquid chromatography: release of amino acids by perfused rat liver.

Perfused rat liver can be considered as one of the most suitable ex vivo models for studies of liver metabolism. To assess the possible effect of L-carnitine and some of its acyl esters on proteolysis in the rat liver, the amino acid derivatization and high-performance liquid chromatographic separation of Tapuhi et al. [Anal. Biochem., 115 (1981) 123] was modified.

ST789: a new synthetic immunomodulator.

ST 789 is a synthetic compound which belongs to a new family of hypoxanthine derivatives exhibiting an aminoacidic function at the N-9 position of the purine ring. Available literature indicates that hypoxanthine derivatives exhibit well established immunomodulant properties. Furthermore, the addition of arginine to these molecules proved able to strongly enhance their immunomodulant activity. We review here the immunomodulant properties of both arginine and arginine-containing compounds, and mostly of ST 789.

Pharmacokinetic investigation of ST 789 in rats and mice.

Pharmacokinetics of ST 789 were investigated in rats and mice after oral, intravenous, subcutaneous and intramuscular routes. A HPLC method validated for pharmacokinetic studies allowed the Authors to assay ST 789 concentration in plasma, urine and tissues. ST 789 interacted poorly with albumin and plasma proteins. Blood-to-plasma concentration ratio proved to range on average from 1.3 to 2.0 in both in vivo and in vitro studies. Plasma concentration-time behaviour after i.v. injection fitted according to the open three-compartment model; after subcutaneous and intramuscular routes two phases were observed and after oral route the absorption and one elimination phases were detected. Pharmacokinetics of ST 789 proved to vary linearly with the dose administered. Cumulative urinary excretion after parenteral administration ranged on average 60-80% and cumulative biliary excretion was 9.47% of the dose given. Oral administration allowed only 2.5% of the drug given to be excreted in urine, this leading to conclude that this drug is poorly absorbed through the intestine wall. After oral administration ST 789 produced relatively high concentration in lungs and lymphatic tissues, this leading to hypothesize a lymphatic component in its enteral absorption.

Immunorestorative properties of ST 789 on experimentally immunosuppressed and infected mice.

ST 789, a newly synthesized chemical characterized by an aminoacidic group joined to the N9 position of the hypoxanthine ring, has recently been shown to be endowed with immunomodulatory properties. In this study we tested ST 789 in vivo for protective effects in Cyclophosphamide-immunosuppressed CD1 mice experimentally infected with several bacterial and fungal pathogens. We found that immunosuppressed mice infected with either fungi or bacteria were significantly protected, as evaluated both by percent mortality and survival time, when treated with doses of ST 789 even as low as 0.2 mg/kg/day. We also observed a marked synergism when the mice were first treated with ST 789 and then additionally treated with subeffective doses of antibiotics such as Amphotericin B, Ceftazidime, and Gentamicin. Even though further studies are required to elucidate the mechanisms underlying the ST 789 effects, these results show that ST 789 is a very promising new immunomodulator whose therapeutic potential has yet to be fully exploited.

Modulation of splenic lymphocyte activities by a new hypoxanthine derivative (ST 789) in immunosuppressed mice.

Splenic lymphocytes of experimentally-immunosuppressed mice of different age (10 weeks and 6 months) were studied to evaluate their functional response following subcutaneous and intraperitoneal treatment with the hypoxanthine derivative N-alpha-5-(1,6-dihydro-6-oxo-9-purinyl)pentyloxy-carbonyl-L- arginine (ST 789). Experimental immunosuppression was carried out by injecting hybrid B6D2F1 mice with a single dose of cyclophosphamide (100 mg/kg, i.p.) 2 hours prior to treatment with ST 789. In the young, we found that ST 789 markedly restored the splenocyte proliferative response, assessed as total amount of 3H-thymidine incorporated by mitogen-stimulated cells. In the adult, however, the ST 789-induced recovery was less pronounced. Finally, the effects of ST 789 treatment on Con A-induced IL-2 production by splenocytes were studied in normal and immunosuppressed mice of 10 weeks.

Effect of calcitonin on different inflammatory models.

The effect of synthetic salmon calcitonin was studied on adjuvant arthritis, pertussis vaccine edema, tuberculin skin reaction, passive direct Arthus reaction and nystatin edema. The results show that calcitonin inhibits these inflammatory processes.