Effects of ST1936, a selective serotonin-6 agonist, on electrical activity of putative mesencephalic dopaminergic neurons in the rat brain.

The serotonin-6 (5-HT6) receptor is the most recently discovered serotonin receptor, and it represents an increasingly promising target for improving cognition in both normal and disease states. Recently, a new selective 5-HT6 receptor agonist, 2-(5 chloro-2-methyl-1H-indol-3-yl)-N,N-dimethylethanamine (ST1936), with nanomolar affinity for 5-HT6 receptors was described. We performed in-vivo electrophysiological studies to investigate the physiological role of 5-HT6 receptors in the control of the function of the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). Extracellular single-unit recordings were performed from putative dopamine-containing neurons in the SNc and VTA of anesthetised rats. In the SNc, acute systemic administration of ST1936 had no effects on basal firing activity of these dopamine neurons; however, in the VTA, ST1936 induced either dose-related increases (45% of cells) or decreases in basal activity of these dopaminergic neurons. Local application of ST1936 into the VTA caused excitation in all of the dopamine neurons, but had no effects on non-dopamine VTA neurons. Both effects of systemic and microiontophoretic ST1936 were completely reversed by the potent and selective 5-HT6 receptor antagonist 5-chloro-N-(4-methoxy-3-piperazin-1-ylphenyl)-3-methyl-2- benzothiophene-sulfonamide (SB271046). Systemic application of another 5-HT6 agonist, 2-(1-{6-chloroimidazo[2,1-b] [1,3]thiazole-5-sulfonyl}-1H-indol-3-yl)ethan-1-amine (WAY-181187), induced dose-dependent inhibition of these VTA dopaminergic neurons. ST1936 and WAY-181187 appear to have different effects on these VTA dopaminergic neurons, potentially due to different mechanisms of action or to the complexity of 5-HT6 receptor functions. Our data demonstrate the need for further investigations into the use of 5-HT6 receptor agonists to control cognitive disfunction, such as in schizophrenia and depression.

Intake of tomato-enriched diet protects from 6-hydroxydopamine-induced degeneration of rat nigral dopaminergic neurons.

There is extensive evidence that oxidative damage of dopamine (DA)-containing neurons in the substantia nigra pars compacta (SNc) may contribute to the pathogenesis of Parkinson's disease (PD). We evaluated the potential neuroprotective effect of diets enriched with wild-type Red Setter (RS) tomato or transgenic High Carotene (HC) tomato, rich in beta-carotene, obtained by the activation of lycopene beta-cyclase (tlcy-b), in an animal model of PD. Male Fischer 344 rats were fed for 14 days with standard Altromin diet, 5% RS- or 5% HC-enriched diet. Seven days after the beginning of this diet regimen, the rats were lesioned by 6-hydroxydopamine (6-OHDA) injected into the left SNc. After further 7 days, the rats were sacrificed, and DA and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in both the left (ipsilateral) and the right (contralateral) striata were measured. Striatal DA levels were reduced by 86.5 +/- 5.0% in control, 86.2 +/- 5.0% in HC-, and 56.0 +/- 9.0% in RS-fed group. Striatal DOPAC was decreased by 85.6 +/- 5.0% in controls, 83.0 +/- 6.0% in HC-, and 58.9 +/- 10.0% in RS-fed group. Blood was obtained from the rats on day 14 and the plasma level of licopene and beta-carotene was measured by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) for the determination of lycopene and beta-carotene levels. The plasma level of lycopene was 4.7 +/- 0.2 ng/ml in 5% RS-fed rats, while it was undetectable (< 2.5 ng ml(-1)) in control and HC-fed rats. The efficacy of RS diet to preserve striatal dopaminergic innervation can be attributed to the ability of lycopene to prevent the degeneration of DA-containing neurons in the SNc.

In vitro and in vivo stability of caffeic acid phenethyl ester, a bioactive compound of propolis.

The in vitro biochemical stability of caffeic acid phenethyl ester in rat and human plasma was investigated and compared with the stability of other caffeic acid esters (chlorogenic acid and rosmarinic acid). The incubation of the compounds in rat plasma for up to 6 h showed that caffeic acid phenethyl ester, but not the other compounds, was hydrolyzed, whereas human plasma did not affect the stability of all the assayed compounds. The products in rat plasma were caffeic acid and an unknown compound, which was identified by mass spectrometry as caffeic acid ethyl ester, produced by transesterification in the presence of ethanol used as vehicle for standard compounds. Specific inhibitors of different plasma esterases allowed the identification of a carboxylesterase as the enzyme involved in the metabolism of caffeic acid phenethyl ester. The oral administration in rats of caffeic acid phenethyl ester in the presence of both ethanol and 2-(2-ethoxyethoxy)ethanol gave rise to a dramatic increase of caffeic acid, as well as low levels of caffeic acid phenethyl ester, caffeic acid ethyl ester, and caffeic acid 2-(2-ethoxyethoxy)ethyl ester, in urine collected within 24 h after treatment. These results suggest that caffeic acid phenethyl ester is hydrolyzed also in vivo to caffeic acid as the major metabolite and that its biological activities should be more properly assayed and compared with those of caffeic acid, its bioactive hydrolysis product. Moreover, alcohols should be carefully used in vivo as solvents for caffeic acid phenethyl ester, since they can give rise to new bioactive caffeic acid esters.

Aspirin protects striatal dopaminergic neurons from neurotoxin-induced degeneration: an in vivo microdialysis study.

The effect of aspirin on dopaminergic neuronal damage induced by in vivo infusion of 1-methyl-4-phenylpiridinium iodide (MPP(+)) and 6-hydroxydopamine (6-OHDA) was studied in rats, using microdialysis. Rat striata were perfused with 1 mM MPP(+) or 6-OHDA for 10 min, causing peak levels of dopamine (DA) in the dialytic fluid, after 40 min. After 24 h, 1 mM MPP(+) was perfused again for 10 min and DA levels measured in the dialytic fluid, as an index of neuronal cell integrity. Pretreatment with Aspidol (lysine acetylsalicylate), 180 mg/kg i.p., 1 h before MPP(+) or 6-OHDA perfusion, did not modify DA extracellular output, on day 1, but restored MPP(+)-induced DA release on day 2, indicating a neuroprotective effect of Aspidol. Conversion of 0.5 mM 4-hydroxybenzoic acid (4-HBA) to 3,4-dihydroxybenzoic acid (3,4-DHBA) was measured as an index of reactive oxygen species (ROS). 6-OHDA, but not MPP(+), significantly enhanced 3,4-DHBA levels in the perfusion fluid. Aspidol (180 mg/kg, i.p.) reduced 6-OHDA-dependent increase of 3,4-DHBA levels. Meloxicam (50 mg/kg, i.p.), a specific cyclooxygenase-2 (COX-2) inhibitor, was ineffective against both neurotoxins. These data suggest that the protective effect of aspirin is due to different mechanisms of action according to the neurotoxin used, and it is independent from COX-2 inhibition.

Caffeic acid phenethyl ester blocks apoptosis induced by low potassium in cerebellar granule cells.

Primary cultures of cerebellar granule neurons (CGNs) were prepared from 8-day-old Wistar rats, and maintained in an appropriate medium containing a high (25 mM) concentration of KCl. To induce apoptosis, culture medium was replaced with serum-free medium (containing 5mM KCl) 8 days after plating. Apoptosis was measured by the terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end-labeling (TUNEL) method, and by flow cytometry. Since there is evidence that an increased formation of reactive oxygen species (ROS) is involved in the apoptosis induced by low K(+) (5mM) concentrations, the potential anti-apoptotic effect of caffeic acid phenethyl ester (CAPE), a potent flavonoid antioxidant, was tested in this experimental model. It was found that CAPE (10 microg/ml) promoted cell survival and was capable of blocking the apoptotic process as assayed by both TUNEL and flow cytometric methods. The same concentration of CAPE prevented the formation of ROS induced by low K(+). Since there is evidence that low K(+)-induced apoptosis in CGNs is associated with a drop in intracellular Ca(2+) concentration ([Ca(2+)](i)), activation of the cell death effector proteases caspase-3 and caspase-9, and of the transcription factor nuclear factor kappa B (NF-kappaB), the interference of CAPE with these purported mediators of apoptosis was also evaluated. It was found that CAPE did not interfere with the marked decrease in [Ca(2+)](i) induced by low K(+), whereas it completely blocked caspase-3, caspase-9, and NF-kappaB activation. It is concluded that CAPE could exert its anti-apoptotic effect in CGNs by blocking ROS formation and by inhibiting caspase activity.

Inhibition of B16-BL6 melanoma lung colonies by semisynthetic sulfaminoheparosan sulfates from E. coli K5 polysaccharide.

Heparin (H), heparan sulfate (HS), and related glycosaminoglycans can inhibit cancer cell invasion, possibly due to their ability to interact with vascular growth factors, adhesion molecules, endoglycosidases, and signaling proteins, in addition to the well-known effects on the clotting system. We evaluated the antitumor activity of a series of semisynthetic sulfaminoheparosan sulfates (SAHSs) with different degree and distribution of sulfates, obtained by chemical modifications of the E. coli K5 polysaccharide, namely type A, B, and C compounds. B16-BL6 melanoma cells (10 5 cells/mouse) were injected intravenously (i.v.) in a lateral tail vein of C57BL6 mice at a dose of 0.5 mg/ mouse together with test compounds. Tumor lung nodules were significantly reduced as compared with controls only by H (95.5 +/- 1.0% inhibition), SAHS-2 (84.2 +/- 5.0% inhibition), and SAHS-4 (91.1 +/- 4.2% inhibition), among compounds tested. SAHS-2 and SAHS-4 are type B compounds, with a sulfate/carboxylate ratio similar to that of H. A typical mammalian HS showed only 54.8% inhibition. Supersulfated low-molecular-weight heparin and heparan sulfate (ssLMWH and ssLMWHS) showed an activity similar to that of unfractionated compounds. H and SAHS-4 inhibited dose dependently B16-BL6 lung colonies, with IC-50 values of 0.05 and 0.1 mg/mouse, respectively. The relationship with ex vivo anticoagulant potency was evaluated by activated partial thromboplastin time (aPTT) on mouse plasma at different time intervals after i.v. injection (0.1 to 0.5 mg/mouse) of the compound. H showed a dose-dependent anticoagulant activity lasting up to 2 hours, whereas SAHS-4 showed a potent anticoagulant effect only at a dose of 0.5 mg/mouse. Accordingly, H but not SAHS-4 consistently inhibited B16-BL6 lung colonies when given 1 hour before tumor cells. SAHS-4 derivatives, with different size and/or affinity depleted of AT binding sites, showed an inhibitory effect on B16-BL6 melanoma similar to that of SAHS-4, suggesting that the greater antitumor effect of H was not due to AT-mediated inhibition of blood clotting. Interactions with other blood inhibitors, such as heparin cofactor II or tissue factor pathway inhibitory protein cannot be ruled out. The better effect of H may be due to persistence in the circulation and/or ability to inhibit tumor neoangiogenesis.