An "ex vivo model" contributing to the diagnosis and evaluation of new drugs in cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. About 2000 mutations have been described so far. We setup an ex vivo model of human nasal epithelial cells (HNECs) to study CF patients testing the effect of novel mutations and molecular therapies. We performed sampling (by brushing), followed by culture and analysis of HNECs using a series of molecular techniques. We performed 50 brushings from CF patients and controls. Using cultured cells, we: i) demonstrated the widely heterogeneous CFTR expression in patients and in controls; ii) defined the splicing effect of a CFTR mutation; iii) assessed the CFTR gating activity in patients bearing different mutations; iv) demonstrated that butyrate significantly enhances CFTR expression. Based on our data, we can conclude: 1) HNEC brushing is performed without anaesthesia and is well tolerated in all CF patients (children and adults); 2) HNECs can be preserved for up to 48 hours before culture allowings multicentre studies; 3) HNECs culture can be considered a suitable model to study the molecular effects of new CFTR gene mutations and/or uncertain meaning specific mutations of carriers; 4) an ex vivo model of HNECs may be used to evaluate, before human use, the effect of new drugs on patients' cells bearing specific CFTR mutations; 5) the methodology is adequate for a quantitative measurement, by fluorescence, of the CFTR gating activity of the HNECs from patients with different genotypes identifying: a) CF patients bearing two severe mutations with an activity < 10% (compared to controls - 100%); b) CF patients bearing at least a mild mutation with an activity of 10-20%; c) CF carriers (heterozygous subjects) with an activity between 40-70%.

Rotura de aneurisma de la arteria pancreaticoduodenal inferior. Manejo endovascular / Ruptured aneurysm of the inferior pancreaticoduodenal artery: Endovascular management

No disponible

Rotura de aneurisma de la arteria pancreaticoduodenal inferior. Manejo endovascular / Uptured aneurysm of the inferior pancreaticoduodenal artery: Endovascular management

No disponible

Bypass aorto-renal con injerto autólogo de arteria femoral superficial en niña con hipertensión renovascular / Renal bypass with autologous superficial femoral artery graft in a child with renovascular hypertension

No disponible

Seudoaneurisma gigante de la arteria mesentérica superior / Giant superior mesenteric artery pseudoaneurysm

No disponible

Preclinical and clinical characterization of the selective 5-HT(1A) receptor antagonist DU-125530 for antidepressant treatment.

BACKGROUND AND PURPOSE: The antidepressant efficacy of selective 5-HT reuptake inhibitors (SSRI) and other 5-HT-enhancing drugs is compromised by a negative feedback mechanism involving 5-HT(1A) autoreceptor activation by the excess 5-HT produced by these drugs in the somatodendritic region of 5-HT neurones. 5-HT(1A) receptor antagonists augment antidepressant-like effects in rodents by preventing this negative feedback, and the mixed ß-adrenoceptor/5-HT(1A) receptor antagonist pindolol improves clinical antidepressant effects by preferentially interacting with 5-HT(1A) autoreceptors. However, it is unclear whether 5-HT(1A) receptor antagonists not discriminating between pre- and post-synaptic 5-HT(1A) receptors would be clinically effective. EXPERIMENTAL APPROACH: We characterized the pharmacological properties of the 5-HT(1A) receptor antagonist DU-125530 using receptor autoradiography, intracerebral microdialysis and electrophysiological recordings. Its capacity to accelerate/enhance the clinical effects of fluoxetine was assessed in a double-blind, randomized, 6 week placebo-controlled trial in 50 patients with major depression (clinicaltrials.gov identifier NCT01119430). KEY RESULTS: DU-125530 showed equal (low nM) potency to displace agonist and antagonist binding to pre- and post-synaptic 5-HT(1A) receptors in rat and human brain. It antagonized suppression of 5-hydroxytryptaminergic activity evoked by 8-OH-DPAT and SSRIs in vivo. DU-125530 augmented SSRI-induced increases in extracellular 5-HT as effectively as in mice lacking 5-HT(1A) receptors, indicating a silent, maximal occupancy of pre-synaptic 5-HT(1A) receptors at the dose used. However, DU-125530 addition to fluoxetine did not accelerate nor augment its antidepressant effects. CONCLUSIONS AND IMPLICATIONS: DU-125530 is an excellent pre- and post-synaptic 5-HT(1A) receptor antagonist. However, blockade of post-synaptic 5- HT(1A) receptors by DU-125530 cancels benefits obtained by enhancing pre-synaptic 5-hydroxytryptaminergic function.

Involvement of 5-HT1A receptors in prefrontal cortex in the modulation of dopaminergic activity: role in atypical antipsychotic action.

Atypical antipsychotics increase dopamine (DA) release in the medial prefrontal cortex (mPFC), an effect possibly involved in the superior effects of atypical versus classical antipsychotics on cognitive/negative symptoms. We examined the role of 5-HT1A receptors in the mPFC on the modulation of dopaminergic activity and the mesocortical DA release in vivo. The highly selective 5-HT1A agonist BAY x 3702 (BAY; 10-40 microg/kg, i.v.) increased the firing rate and burst firing of DA neurons in the ventral tegmental area (VTA) and DA release in the VTA and mPFC. The increase in DA release in both areas was potentiated by nomifensine coperfusion. The selective 5-HT1A antagonist WAY-100635 reversed the effects of BAY in both areas, and the changes in the VTA were prevented by frontocortical transection. The application of BAY in rat and mouse mPFC by reverse dialysis increased local extracellular DA at a low concentration (3 microM) and reduced it at a higher concentration (30 microM). Both effects disappeared in 5-HT1A knock-out mice. In the presence of bicuculline, BAY reduced DA release at all concentrations. The atypical antipsychotics clozapine, olanzapine, and ziprasidone (but not haloperidol) enhanced DA release in the mPFC of wild-type but not 5-HT1A knock-out mice after systemic and local (clozapine and olanzapine) administration in the mPFC. Likewise, bicuculline coperfusion prevented the elevation of DA release produced by local clozapine or olanzapine application. These results suggest that the activation of mPFC 5-HT1A receptors enhances the activity of VTA DA neurons and mesocortical DA release. This mechanism may be involved in the elevation of extracellular DA produced by atypical antipsychotics.

The activation of 5-HT receptors in prefrontal cortex enhances dopaminergic activity.

Atypical antipsychotics show preferential 5-HT 2A versus dopamine (DA) D2 receptor affinity. At clinical doses, they fully occupy cortical 5-HT2 receptors, which suggests a strong relationship with their therapeutic action. Half of the pyramidal neurones in the medial prefrontal cortex (mPFC) express 5-HT 2A receptors. Also, neurones excited through 5-HT 2A receptors project to the ventral tegmental area (VTA). We therefore hypothesized that prefrontal 5-HT 2A receptors can modulate DA transmission through excitatory mPFC-VTA inputs. In this study we used single unit recordings to examine the responses of DA neurones to local (in the mPFC) and systemic administration of the 5-HT 2A/2C agonist 1-[2,5-dimethoxy-4-iodophenyl-2-aminopropane] (DOI). Likewise, using microdialysis, we examined DA release in the mPFC and VTA (single/dual probe) in response to prefrontal and systemic drug administration. The local (in the mPFC) and systemic administration of DOI increased the firing rate and burst firing of DA neurones and DA release in the VTA and mPFC. The increase in VTA DA release was mimicked by the electrical stimulation of the mPFC. The effects of DOI were reversed by M100907 and ritanserin. These results indicate that the activity of VTA DA neurones is under the excitatory control of 5-HT 2A receptors in the mPFC. These observations may help in the understanding of the therapeutic action of atypical antipsychotics.

Desensitization of 5-HT(1A) autoreceptors by a low chronic fluoxetine dose effect of the concurrent administration of WAY-100635.

Using microdialysis, receptor autoradiography and in situ hybridization, we examined the effects of fluoxetine alone or with WAY-100635 on: (a) extracellular 5-HT in frontal cortex; and (b) density and sensitivity of 5-HT(1A) autoreceptors in rat brain. WAY-100635 (0.3 mg/kg, s.c.) doubled the increase in extracellular 5-HT produced by fluoxetine (3 mg/kg, i.p.) in frontal cortex. Two-week minipump treatments with these daily doses significantly raised extracellular 5-HT to 275 +/- 33% (fluoxetine) and 245 +/- 10% (fluoxetine plus WAY-100635) of controls. Fluoxetine 3 mg/kg.day desensitized dorsal raphe 5-HT(1A) autoreceptors, an effect prevented by the concurrent WAY-100635 administration. However, WAY-100635 (alone or with fluoxetine) did not change 5-HT(1A) autoreceptor sensitivity. The density of 5-HT(1A) receptors and its encoding mRNA, was unaffected by these treatments. These results suggest that prolonged blockade of 5-HT(1A) receptors in vivo prevents the autoreceptor desensitization induced by fluoxetine but does not result in receptor sensitization.

Effects of chronic oestrogen treatment are not selective for uterine noradrenaline-containing sympathetic nerves: a transplantation study.

Previous studies have shown that chronic administration of oestrogen during postnatal rat development dramatically reduces the total content of noradrenaline in the uterine horn, abolishes myometrial noradrenergic innervation and reduces noradrenaline-fluorescence intensity of intrauterine perivascular nerve fibres. In the present study we analysed if this response is due to a direct and selective effect of oestrogen on the uterine noradrenaline-containing sympathetic nerves, using the in oculo transplantation method. Small pieces of myometrium from prepubertal rats were transplanted into the anterior eye chamber of adult ovariectomised host rats. The effect of systemic chronic oestrogen treatment on the reinnervation of the transplants by noradrenaline-containing sympathetic fibres from the superior cervical ganglion was analysed on cryostat tissue sections processed by the glyoxylic acid technique. In addition, the innervation of the host iris was assessed histochemically and biochemically. The histology of the transplants and irises was examined in toluidine blue-stained semithin sections. These studies showed that after 5 wk in oculo, the overall size of the oestrogen-treated transplants was substantially larger than controls, and histology showed that this change was related to an increase in the size and number of smooth muscle cells within the transplant. Chronic oestrogen treatment did not provoke trophic changes in the irideal muscle. Histochemistry showed that control transplants had a rich noradrenergic innervation, associated with both myometrium and blood vessels. Conversely, in oestrogen-treated transplants only occasional fibres were recognised, showing a reduced NA fluorescence intensity. No changes in the pattern and density of innervation or in the total content of noradrenaline of the host irises were detected after chronic exposure to oestrogen. We interpreted these results to indicate that the effects of oestrogen on uterine noradrenaline-containing sympathetic nerves are neither selective or direct, but result from an interaction between sympathetic nerve fibres with the oestradiol-primed uterine tissue. A potential effect of oestrogen on the neurotrophic capacity of the uterus is discussed.

Differential effects of prepubertal chronic oestrogen treatment on the synthesis of noradrenaline in uterine myometrial and perivascular sympathetic nerves.

Previous studies have shown that chronic administration of oestrogen to prepubertal rats reduces the total content of noradrenaline in the uterine horn, abolishes myometrial noradrenergic innervation and reduces noradrenaline-fluorescence intensity of intrauterine perivascular nerve fibres. The mechanisms underlying these changes are not known. In the present study we have analysed the effects of prepubertal chronic oestrogen treatment on the synthesis of noradrenaline in the rat uterine sympathetic nerves using biochemical and immunohistochemical approaches. Tyrosine hydroxylase activity was evaluated biochemically, by measuring the in vivo accumulation of dihydroxyphenylalanine (DOPA) in the presence of a DOPA-decarboxylase inhibitor. In addition, nerve fibres were visualised immunohistochemically using antibodies against tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DbetaH) and the general marker protein gene product 9.5 (PGP 9.5). After chronic oestrogen treatment, the total content of noradrenaline of the uterine horn was reduced, whereas the total content of DOPA was increased. In controls, TH-immunoreactive, DbetaH-immunoreactive and PGP 9.5-immunoreactive nerve fibres were distributed in both the circular and longitudinal myometrial layers and in the blood vessels of the intran-myometrial region. After chronic oestrogen treatment the only fibres recognised by the three antibodies were those associated with the blood vessels, but no myometrial-associated fibres could be recognised. These results suggest that noradrenaline synthesis is selectively reduced in myometrial-associated uterine sympathetic nerves, but is preserved in perivascular sympathetic nerves. The increased DOPA levels measured after chronic exposure to oestrogen was interpreted as the consequence of the substantial increase in size and number of blood vessels observed in the uterus of oestrogen-treated animals. A possible neurodegenerative effect of oestrogen on myometrial sympathetic fibres is discussed.

Dopaminergic pharmacology and antioxidant properties of pukateine, a natural product lead for the design of agents increasing dopamine neurotransmission.

The dopaminergic and antioxidant properties of pukateine [(R)-11-hydroxy-1,2-methylenedioxyaporphine, PUK], a natural aporphine derivative, were analyzed in the rat central nervous system. At dopamine (DA) D1 ([3H]-SCH 23390) and D2 ([3H]-raclopride) binding sites, PUK showed IC50 values in the submicromolar range (0.4 and 0.6 microM, respectively). When the uptake of tritiated dopamine was assayed by using a synaptosomal preparation, PUK showed an IC50 = 46 microM. In 6-hydroxydopamine unilaterally denervated rats, PUK (8 mg/kg but not 4 mg/kg) elicited a significant contralateral circling, a behavior classically associated with a dopaminergic agonist action. When perfused through a microdialysis probe inserted into the striatum, PUK (340 microM) induced a significant increase in dopamine levels. In vitro experiments with a crude rat brain mitochondrial suspension showed that PUK did not affect monoamine oxidase activities, at concentrations as high as 100 microM. PUK potently (IC50 = 15 microM) and dose-dependently inhibited the basal lipid peroxidation of a rat brain membrane preparation. As a whole, PUK showed a unique profile of action, comprising an increase in extracellular DA, an agonist-like interaction with DA receptors, and antioxidant activity. Thus, PUK may be taken as a lead compound for the development of novel therapeutic strategies for Parkinson disease.

Monoamine oxidase inhibitory properties of some methoxylated and alkylthio amphetamine derivatives: structure-activity relationships.

The monoamine oxidase (MAO) inhibitory properties of a series of amphetamine derivatives with different substituents at or around the para position of the aromatic ring were evaluated. In in vitro studies in which a crude rat brain mitochondrial suspension was used as the source of MAO, several compounds showed a strong (IC50 in the submicromolar range), selective, reversible, time-independent, and concentration-related inhibition of MAO-A. After i.p. injection, the compounds induced an increase of serotonin and a decrease of 5-hydroxyindoleacetic acid in the raphe nuclei and hippocampus, confirming the in vitro results. The analysis of structure-activity relationships indicates that: molecules with amphetamine-like structure and different substitutions on the aromatic ring are potentially MAO-A inhibitors; substituents at different positions of the aromatic ring modify the potency but have little influence on the selectivity; substituents at the para position such as amino, alkoxyl, halogens, or alkylthio produce a significant increase in the activity; the para-substituent must be an electron donor; bulky groups next to the para substituent lead to a decrease in the activity; substituents located at positions more distant on the aromatic ring have less influence and, even when the substituent is a halogen (Cl, Br), an increase in the activity of the compound is obtained. Finally, the MAO-A inhibitory properties of some of the compounds evaluated are discussed in relation to: (a) potential antidepressant activity, and (b) their reported hallucinogenic, neurotoxic, or anxiolytic effects.

Behavioral effects of the putative anxiolytic (+/-)-1-(2,5-dimethoxy-4-ethylthiophenyl)-2-aminopropane (ALEPH-2) in rats and mice.

Behavioral effects of the phenethylamine derivative (+/-)-1-(2,5-dimethoxy-4-ethylthiophenyl)-2-aminopropane (ALEPH-2) were studied in mice and rats. Murine locomotor activity, measured with a photocell actometer, was markedly depressed following IP injection of 2 and 6 mg/kg of the drug. The same doses of the drug also decreased frequency and duration of head dipping and the number of rearings in the hole board apparatus. In the murine elevated plus maze 2 and 6 mg/kg of ALEPH-2 increased the percentage of both open arm entries and time. The total number of entries into the enclosed arms was not significantly affected by the drug. In the rat, 2-12 mg/kg ALEPH-2, IP, decreased photobeam counts in the actometer in a dose-dependent fashion. Both 2 and 4 mg/kg of the drug increased the percentage of open arm entries, but only the highest dose significantly increased the percentage of time spent on the open arms. The dose of 4 mg/kg ALEPH-2 also significantly decreased the total number of enclosed arm entries. Finally, in a recently developed model of anxiety and memory, the elevated T-maze, the doses of 2 and 4 mg/kg ALEPH-2 did not change inhibitory avoidance of the open arms. Nevertheless, the highest dose had an amnestic effect on this task, repeated 72 h later in the absence of drug. In addition, this dose significantly increased the latency to escape from the open arms and had an amnestic effect measured 72 h later. Overall, these results indicate that ALEPH-2 possesses anxiolytic, amnestic as well as sedative and/or motor depressant actions.

Monoamine oxidase inhibitory effects of some 4-aminophenethylamine derivatives.

The in vitro and ex vivo monoamine oxidase (MAO) inhibitory effects of (+/-)4-dimethylamino-alpha-methyl-phenethylamine (4-DMAA) and (+/-)4-methylamino-alpha-methyl-phenethylamine (4-MAA) were reassessed, in comparison with the previously unstudied achiral parent compound, 4-dimethyl-aminophenethylamine (4-DMAPEA) and with a salt of 4-DMAA enriched in the levo isomer, ("-")-4-DMAA, using amiflamine [S-(+)-4-dimethylamino-alpha,2-dimethylphenethylamine] as positive control. The in vitro studies confirmed that 4-amino-alpha-methylphenethylamine derivatives are highly selective and reversible MAO-A inhibitors. Furthermore, ("-")-4DMAA was less active than the racemic mixture. The side chain-unsubstituted compound, 4-DMAPEA, proved to be a nonselective and reversible MAO inhibitor. The ex vivo results, in which catecholamines, serotonin (5-HT) and their metabolites were measured in two brain regions after i.p. administration, confirmed the results obtained in vitro. These results are consistent with the suggestion that the 4-amino group contributes to MAO inhibitory effects of alpha-methyl-phenethylamines, and show that the presence and orientation of an alpha-methyl side chain substituent may be important when determining the potency and selectivity of these compounds. All compounds tested could be quantified by HPLC with electrochemical detection.

4-Dimethylaminophenethylamine, a sensitive, specific, electrochemically detectable monoamine oxidase-B substrate.

4-Dimethylaminophenethylamine (DMAPEA) was characterized as an MAO substrate. This compound was unaffected by MAO-A, while its oxidation by MAO-B was linear as a function of both time and enzyme concentration, with Km = 5.8 microM and Vmax = 21.2 pmol/min/mg protein, using a crude rat brain mitochondrial suspension as source of MAO. Both DMAPEA and its oxidation product, 4-dimethylaminophenylacetic acid (DMAPAA), can be detected electrochemically at 0.85 V. The high MAO-B affinity and selectivity of DMAPEA, together with its low oxidation potential, make this molecule a unique tool to determine MAO-B activity in a wide variety of tissue preparations using HPLC-ED.