Polyunsaturated fatty acid levels in rat tissues after chronic treatment with dietetic oils.

BACKGROUND: The essential fatty acids can be helpful in the prevention of several pathologies. The purpose of this study was to quantify the major n-3 and n-6 fatty acids in tissues of rats fed with flaxseed oil and with a dietetic oil in order to evaluate how their chronic supplementation could influence the correspondent in vivo levels and to study the effectiveness of the dietetic oil compared to flaxseed oil. RESULTS: Fatty acids were successfully extracted from biological samples, subjected to derivatization procedure and analysed by high-performance liquid chromatography with UV detection under gradient elution mode. The developed method showed good linearity, precision and accuracy, with recoveries ranging from 89% to 92%. Animals treated with flaxseed and dietetic oils showed enhanced levels of n-3 fatty acids compared to control groups, with significantly higher levels of eicosapentaenoic acid and docosahexaenoic acid in the brain and in the adipose tissue of the dietetic group compared to the flaxseed group. CONCLUSION: The obtained data underline that the tested oils can effectively enhance the tissue levels of n-3 fatty acids and therefore they could be successfully used in the dietetic treatment of lipid-related diseases.

Further evidence of the antiulcer activity of IAC, a novel free radical scavenger.

It has been proposed that free radicals, reactive oxygen species (ROS) and reactive nitrogen species play a critical role in gastric mucosal damage. It is well known that the exposure of gastric mucosa to damaging factors such as stress and nonsteroidal anti-inflammatory drugs produces acute ulcers that are mainly mediated by ROS. The aim of the present study was to investigate the gastroprotective properties of bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate (IAC), a novel nonpeptidyl low-molecular-weight radical scavenger, in two different models of gastric ulcer in rats caused by ROS. IAC was orally administered at the doses of 50 and 100 mg/kg before gastric ulceration induced by indomethacin and water immersion and restraint stress. The number and severity of gastric lesions, following macroscopic inspection of the mucosa, were evaluated and expressed as an ulcer score. Oral administration of IAC dosed at 50 and 100 mg/kg was able to significantly prevent gastric ulceration induced by indomethacin and by stress. The gastroprotective effect of IAC on gastric mucosa could be attributed to its intrinsic antioxidant activity, suggesting it as a novel antiulcer agent.

A novel class of allosteric modulators of AMPA/Kainate receptors.

The rapid hydrolysis in vivo of IDRA21 to 2-amino-5-chlorobenzensulfonamide has been demonstrated by microdialysis experiments. The IDRA21 metabolite possess in vitro a biological activity similar to that of IDRA21 itself. Taking 2-amino-5-chlorobenzensulfonamide as lead compound, a novel class of AMPAR positive allosteric modulators has been prepared.

Anti-ulcer activity of IAC, a novel free-radical scavenger, in rats.

OBJECTIVES: We investigated the ability of a novel low-molecular-weight free-radical scavenger, bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)-decandioate (IAC), to protect the gastric mucosa from indometacin-induced ulceration. METHODS: The pharmacological effects of IAC, administered orally or by intraperitoneal injection, on the gastric mucosa were assessed in a rat model of gastric ulceration induced by indometacin. The effect of IAC on the level of prostaglandin PGE2 in the gastric mucosa was also investigated. RESULTS: IAC, which has no ulcerative activity per se, had a preventive and protective activity against indometacin-induced gastric ulceration. This effect could be only partially attributed to a modulatory effect of IAC on PGE2 levels; it is more likely to be due to the antioxidant activity of the compound. CONCLUSIONS: Taking into account the properties of IAC and the mechanisms underlying gastric inflammation elicited by non-steroidal anti-inflammatory drugs, IAC may represent a novel anti-ulcer agent.

Improved sexual behavior in male rats treated with a Chinese herbal extract: hormonal and neuronal implications.

AIM: To investigate the influence of an extract obtained from five Chinese medicinal plants on sexual behavior of adult male rats. METHODS: The extract was administered at doses of 30, 60 and 120 mg/kg by oral gavage, acutely (one time, 45 min before mating test) or subchronically (daily for 10 days) in sexually potent and sexually sluggish/impotent rats. Sexual behavior, serum levels of luteinizing hormone (LH) and testosterone (T) were evaluated in treated rats and compared with controls receiving vehicle. The effect of the extract on central dopaminergic neurotransmission was assessed in the nucleus accumbens using a microdialysis technique. RESULTS: In sexually potent rats, both acute and subchronic treatment with the extract dosed at 30 and 60 mg/kg reduced mount latency and intromission latency. In sluggish/impotent rats, the acutely administered extract at the dose of 60 mg/kg shortened ejaculation latency, whereas subchronically administered at the doses of 30 and 60 mg/kg, reduced mount, intromission and ejaculation latencies, increasing also the percentage of mounting and ejaculating rats. The extract dosed at 60 mg/kg significantly increased LH and T following acute and subchronic administration and increased 3,4-dihydroxyphenylacetic acid levels in the nucleus accumbens, 30 min after the acute administration. CONCLUSION: The improvement in both appetitive and consummatory components of sexual behavior observed in male rats treated with the extract could be ascribed to increased serum T level in parallel with the activation of the central dopaminergic system.

Translocator protein (18kDa): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function.

The peripheral-type benzodiazepine receptor or recognition site (PBR) is a widely distributed transmembrane protein that is located mainly in the outer mitochondrial membrane. The PBR binds to high-affinity drug ligands and cholesterol. Many functions are associated directly or indirectly with the PBR, including the regulation of cholesterol transport and the synthesis of steroid hormones, porphyrin transport and heme synthesis, apoptosis, cell proliferation, anion transport, regulation of mitochondrial functions and immunomodulation. Based on these functions, there are many potential clinical applications of PBR modulation, such as in oncologic, endocrine, neuropsychiatric and neurodegenerative diseases. Although "PBR" is a widely used and accepted name in the scientific community, recent data regarding the structure and molecular function of this protein increasingly support renaming it to represent more accurately its subcellular role (or roles) and putative tissue-specific function (or functions). Translocator protein (18kDa) is proposed as a new name, regardless of the subcellular localization of the protein.

Evidence that natural benzodiazepine-like compounds increase during spontaneous labour.

Natural benzodiazepine-like compounds (NBDZ) are present in the blood of normal people free of commercial benzodiazepine medication. In this work, we evaluated the levels of NBDZ in maternal/foetal serum during delivery after spontaneous labour (VD) or caesarean section (CS). For both the VD (n=11) and the CS (n=11) groups (VD+CS=22), three blood samples were collected at three different times: the first was collected three days before labour, the second immediately after delivery or at fetal abdominal extraction and the third one was obtained at second day post-partum. NBDZ were measured by radioreceptor binding assay after HPLC extraction and purification while cortisol was measured through radioimmunoassay. In the VD group, a significant increase of NBDZ levels occurred at labour in comparison with the levels found in pre- and post-partum periods. By the contrary, no differences in NBDZ levels were found in the CS group at the three different times. The levels of cortisol in the VD group were found to be higher at labour than that determined at pre- and post-partum. Again no significant changes were found in the CS group. These findings suggest for the first time that labour is associated with a marked increase of NBDZ which could be envisaged as a stress-related event.

Endozepines in recurrent stupor.

Stupor is a condition from which the subject can be aroused only by vigorous stimuli. Most patients with stupor have a diffuse organic cerebral dysfunction. Rarely stupor is recurrent and no specific causes can be found. Patients with idiopathic recurrent stupor were awakened by i.v. administration of an antagonist (flumazenil) of the benzodiazepine recognition site located in the GABA(A) receptor. Since no exogenous benzodiazepines were detected in plasma and cerebrospinal fluid by high performance liquid chromatography, an excess of endogenous benzodiazepine-like compounds (endozepines) was proposed as the cause of stupor. The existence of endozepines, their widespread distribution in the CNS and their involvement in hepatic encephalopathy are established. However, the origin of these compounds, how biosynthesis occurs and the mechanisms and causes through which they alter brain functions are poorly understood. The fact that a number of synthetic benzodiazepines are difficult to detect using conventional techniques and the discovery that some cases of recurrent stupor were caused by fraudulent administration of lorazepam question whether the concept of endozepine recurrent stupor can be sustained. This review summarizes the state of endozepine physiology and pharmacology and the clinical syndromes attributed to their involvement. A diagnostic work-up to define endozepine-induced recurrent stupor is suggested.

Ferula hermonis impairs sexual behavior in hormone-primed female rats.

The influence of Ferula hermonis root extract on sexual behavior was studied in female rats. Sexual receptivity, proceptivity and paced mating behavior were evaluated in ovariectomized females primed with estradiol benzoate (EB) and progesterone (P) and then treated with F. hermonis extract acutely (30 and 60 mg/kg) or subchronically (1 and 10 mg/kg daily for 10 consecutive days). A significant reduction in lordosis responses was observed in rats after acute (60 mg/kg) or subchronic (1 and 10 mg/kg) administration of the plant extract. Similarly, a decrease in proceptive behaviors was exhibited by F. hermonis treated rats in comparison with EB+P controls. No difference was found in the patterns of paced mating behavior between control and treated animals. The present results demonstrate that the acute or repeated ingestion of F. hermonis specifically impairs the receptive and proceptive components of female sexual behavior. The effect could be the consequence of an antiestrogenic action of the extract in hormone-primed female rats.

Synthesis and antiplatelet activity of thioaryloxyacids analogues of clofibric acid.

The thiophene-, benzothiazole- and pyridine-thioaryloxyacids analogues of clofibric acid were synthesized and their antiplatelet activity was screened. Some compounds exhibited antiaggregating properties. The platelet-related haemostasis was measured on a PFA-100 analyzer using bull blood.

2,1,3-Benzothiadiazine derivatives: synthesis and screening versus PDE4 enzyme.

A series of N-1,3 disubstituted 2,1,3-benzothiadiazine derivatives (BTDs) were synthesized and evaluated for their inhibitory activity versus enzymatic isoform PDE4 extracted from U937 cell line. Some of the tested compounds showed a high PDE4 inhibitory activity at 100 microM and the IC(50) value of the most interesting terms were evaluated. The structure-activity relationships of these compounds showed that the 3,5-di-tert-butyl-4-hydroxybenzyl moiety at N-1 position is important to obtain activity at micromolar level as previously reported. For the same compounds the antioxidant activity were evaluated highlighting 14 as the most significative one. The introduction of other bulky substituents in N-1 position is detrimental.

IDRA-21, a positive AMPA receptor modulator, inhibits synaptic and extrasynaptic NMDA receptor mediated events in cultured cerebellar granule cells.

IDRA-21 (7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide) reduces alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) receptors desensitisation in vitro and restores learning and cognitive impairment in vivo. In this study, we show that in cerebellar granule cells (CGCs) in culture IDRA-21 reduces N-methyl-d-aspartate receptor (NMDAR) whole-cell currents. The effect is neither competitive nor voltage-dependent. The reduction of NMDA currents is stronger at low glycine concentrations suggesting an interaction with this site. IDRA-21 shortens miniature excitatory postsynaptic currents mediated by NMDARs (NMDA-mEPSCs) in CGCs grown in low potassium with no effect on peak amplitudes. By using fast glutamate application onto CGCs nucleated patches, we found that IDRA-21 decreases both decay time constant and amplitude of the current. Experiments performed on recombinant NMDAR expressed in HEK 293 cells showed that IDRA-21 was more effective on NR1a-NR2B than NR1a-NR2A receptors highlighting a subunit selectivity of the drug. Our findings make light on a novel target for IDRA-21: NMDA receptors function is negatively modulated and the different action at the level of extrasynaptic and synaptic receptors could be ascribed to a partial selectivity for NR2B subunits.

Synthesis of 3,4-dihydro-2H-1,2,4-benzo-thiadiazine 1,1-dioxide derivatives as potential allosteric modulators of AMPA/kainate receptors.

A series of 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide derivatives were synthesized and evaluated for their activity as allosteric modulators of kainate-activated currents in primary cultures of cerebellar granule neurons. Substitution of different groups at the 3-position of the benzothiadiazine ring distinguished between positive and negative allosteric modulatory properties.

Cognitive deficits and changes in gene expression of NMDA receptors after prenatal methylmercury exposure.

Previous studies showed learning and memory deficit in adult rats that were prenatally exposed to methylmercury chloride (MMC) in an advanced stage of pregnancy (15 days). Under these conditions, the cognitive deficits found at 60 days of age paralleled particularly changes in the N-methyl-D-aspartate (NMDA) receptor characteristics. In the present study, we report the behavioral effects of a single oral dose of MMC (8 mg/kg) administered earlier at gestational day 8. The use of different learning and memory tests (passive avoidance, object recognition, water maze) showed a general cognitive impairment in the in utero-exposed rats tested at 60 days of age compared with matched controls. Considering the importance of the glutamatergic receptor system and its endogenous ligands in learning and memory process regulation, we surmised that MMC could affect the gene expression of NMDA receptor subtypes. The use of a sensitive RNase protection assay allowed the evaluation of gene expression of two families of NMDA receptors (NR-1 and NR-2 subtypes). The result obtained in 60-day-old rats prenatally exposed to MMC, showed increased mRNA levels of the NR-2B subunit in the hippocampus but not in the frontal cortex. The data suggest that the behavioral abnormalities of MMC-exposed rats might be ascribed to a neurotoxic effect of the metal that alters the gene expression of a specific NMDA receptor subunit in the hippocampus.

Apigenin modulates GABAergic and glutamatergic transmission in cultured cortical neurons.

Using the patch-clamp technique, we studied the modulation of ionotropic gamma-aminobutyric acid (GABA) and glutamate neurotransmission by apigenin, a flavonoid with sedative and antidepressant activity. Apigenin reversibly reduced GABA-evoked currents mediated by alpha1beta2gamma2 receptors expressed in HEK293 cells. Amplitude and frequency of spontaneous postsynaptic inhibitory currents (sIPSCs) mediated by GABA(A) receptors were also decreased by apigenin in cultured cortical neurons. The flavonoid was almost inactive on alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) mediated currents while it reduced N-methyl-D-aspartate (NMDA) receptor mediated responses with a half maximal inhibiting concentration (IC50) of 10 microM. The flavonoid inhibited also peak amplitude and frequency of spontaneous postsynaptic excitatory currents (sEPSCs). Finally, apigenin is neuroprotective against glutamate-induced neurotoxicity in cerebellar and cortical neurons in culture. Our data reveal the antagonistic effect of apigenin on GABA and NMDA channels. While the inhibition on GABA receptor cannot explain the effects of the drug in vivo our data on NMDA channels reveal a new target of apigenin. A reduction of the network excitability could thus account for the sedative effects. Furthermore, our data suggest a potential neuroprotective activity of apigenin.

Morphological and receptorial changes in the epididymal adipose tissue of rats subjected to a stressful stimulus.

Obesity is nowadays related to other pathological conditions such as inflammation, insulin resistance, and diabetes, but little is known about the relationship between psychological stress and adipocytes. We decided to study the expression of the translocator protein (TSPO) 18-kDa, peroxisome proliferator-activated receptor-γ (PPAR-γ), mitochondrial uncoupling protein-1 (UCP-1), and adipocyte morphology in the adipose tissue of rats subjected to stress conditions. In our model of stress, rats fasted for 24 h were placed in a restraint cage and then immersed vertically to the level of the xiphoid process in a water bath at 23 °C for 7 h. After that period, we removed the epididymal adipose tissues for the subsequent analysis. The optical and electron microscopy revealed that adipocytes of control rats formed a continuous epithelial-like cell layer; on the contrary in the adipocytes of stressed rats some cells have merged together and the number of vessels formed seems to increase. Stressed adipocytes presented unilocular cells with numerous mitochondria with a morphology ranging between that of brown adipose tissue (BAT) and white adipose tissue (WAT). Interestingly, when we investigated the subcellular distribution of UCP-1 by immunogold electron microscopy, the adipose tissue of stressed rats was positive for UCP-1. From the immunoblot analysis with anti-PPAR-γ antibody, we observed an increased expression of PPAR-γ in the adipocytes of stressed group compared with control group (P < 0.05). Stress induced the expression of TSPO 18-kDa receptor (B(max) = 106.45 ± 5.87 fmol/mg proteins), which is undetectable by saturation-binding assay with [(3)H]PK 11195 in the control group.

Anti-inflammatory activity of the non-peptidyl low molecular weight radical scavenger IAC in carrageenan-induced oedema in rats.

OBJECTIVE: In this research we investigated the anti-inflammatory activity of a non-peptidyl low molecular weight radical scavenger (IAC) in an acute and chronic animal model of inflammation. METHODS: For this purpose the effect of IAC (10, 25, 50 mg/kg) was tested in rats on the associated behavioral responses to subsequent inflammatory and noxious challenges, such as hind paw oedema induced by intra-plantar injection of carrageenan and granuloma induced by subcutaneous implant of a cotton pellet, using indometacin (2.5 mg/kg) as reference drug. Moreover, the serum level of several cytokines was tested in the animal treated (or not) with IAC (50 mg/kg) both in the absence and presence of carrageenan-induced inflammation. KEY FINDINGS: IAC showed a significant anti-inflammatory activity in both in acute and chronic models of inflammation. In addition IAC down regulated significantly the serum levels of interleukin (IL) 2 and IL6 whereas it increased the serum concentration of IL1α and glutathione. CONCLUSION: Although it remains to be elucidated whether or not the antioxidant property of IAC is directly responsible for the modulation of the tested cytokines, these results suggest IAC to be a possible candidate for a novel anti-inflammatory compound.

Quantitative analysis of acetylcholine in rat brain microdialysates by liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A liquid chromatography tandem mass spectrometry (LC/MS/MS) method has been developed for the quantitative analysis of acetylcholine in rat brain dialysates. The separation of acetylcholine (ACh), choline (Ch), acetyl-ß-methylcholine (IS) from endogenous compounds and Ringer's salts was achieved with cation exchange chromatography. Optimization of chromatographic and mass spectrometry parameters were perfomed in order to improve sensitivity of the method. The limit of detection were 0.05 and 3.75 fmol on column with S/N ratio of 3:1 for ACh and Ch, respectively. The limit of quantitation (LOQ) for ACh and Ch measured in Ringer's solution were 0.05 nM (0.25 fmol) and 3.75 nM (18.75 fmol), respectively at S/N ratio of 10:1. Linearity of the method has been evaluated in the concentrations range between 0.05 and 5.00 nM and 3.75 and 200 nM for ACh and Ch respectively. The correlation coefficients were 0.999 and 0.995 for ACh and Ch respectively, indicating very good linearity. The LC/MS/MS method developed has been applied to evaluate the effect of oral administration of 7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide (IDRA21), a positive modulators of AMPA receptor, on the release of ACh in the rat prefrontal cortex by microdialysis.

The phytoestrogen ferutinin affects female sexual behavior modulating ERalpha expression in the hypothalamus.

This study was designed to assess the effect of the phytoestrogenic compound ferutinin, chronically administered in ovariectomized progesterone primed rats, alone or in combination with estradiol benzoate. After 2, 3 and 4 weeks of treatments, female rats were tested for receptive (lordosis) and proceptive behaviors (hops, darts and ear wigglings). Ferutinin given alone markedly increased the intensity of the lordotic response in ovariectomized rats but failed to significantly affect proceptivity. On the other hand estradiol benzoate significantly increased both receptive and proceptive behaviors. When administered in combination with estradiol, ferutinin reduced the increase in receptivity and proceptivity due to estrogen effects, acting as an antiestrogen. At the end of the behavioral experiments, animals were sacrificed and Western blot analysis of estrogen receptor alpha (ERalpha) levels was performed in the dissected hypothalami. Ferutinin increased ERalpha expression when administered alone, as estradiol did, but decreased the response to estradiol when administered in combination. These results suggest that ferutinin displays estrogenic or antiestrogenic activity through ERalpha in the hypothalamus, depending on the absence or the presence of estrogen priming.

Peripheral benzodiazepine receptor (PBR) new insight in cell proliferation and cell differentiation review.

The peripheral benzodiazepine receptor (PBR), is an 18 kDa protein of the mammalian mitochondrial membrane and is a highly conserved protein among the mammalian. PBR is involved in numerous biological functions, including steroid biosynthesis, mitochondrial oxidative phosporylation and cell proliferation. The presence of PBR at the nuclear subcellular level has been demonstrated in aggressive breast cancer cell line and human glioma cells, where it seems to be involved in cell proliferation. In our previous studies we investigated the presence of nuclear PBR in different hepatic tumour cell lines with regard to binding to [3H] PK 11,195 and protein analysis. The results obtained by saturation binding experiments and Scatchard analysis of nuclear PBR density in parallel with the results on the growth curves of the cell lines tested, indicate that the nuclear PBR density correlates inversely with cell doubling time. Moreover, the cell line with high nuclear PBR proliferates in response to PBR ligands, whereas that with low nuclear PBR does not. All these findings support the idea that PBR could play a pivotal role in cell proliferation and this receptor protein could be potentially important either in early diagnosis or chemopreventive strategies against degenerative disease.