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M Beltramo

Publications and source records attributed to M Beltramo.

At least 19 recordsLinked to original sources
PubMed2006

CB2 receptor-mediated antihyperalgesia: possible direct involvement of neural mechanisms.

In mouse the cannabinoid receptor 2 (CB2) agonists L768242 and (+)-AM1241, at doses of 30 mg/kg i.p. and 1 and 3 mg/kg i.v., respectively, reduced the second phase of nocifensive behaviors elicited by formalin intraplantar injection. This effect was counteracted by the selective CB2 antagonist SR144528 (1 mg/kg i.p.). In rat (+)-AM1241 (3 and 6 mg/kg i.v.) and L768242 (30 mg/kg i.p.) reduced allodynia elicited by L5-L6 spinal nerve ligation. SR144528 reverted these effects, supporting a CB2-mediated action. To clarify the mechanisms underlying these effects we investigated CB2 gene expression and function in the nervous system. CB2 mRNA was expressed in spinal cord and dorsal root ganglia (DRG) of both sham and neuropathic rats and was up-regulated in the ipsilateral spinal cord of neuropathic rats. Expression studies demonstrated the presence of CB2 mRNA in culture of spinal cord microglia. A biomarker, CGRP, was used to investigate modulation of DRG primary afferents by CB2 agonists. Both L768242 and (+)-AM1241 dose dependently (EC50 of 3.6 and 4.5 nM, respectively) reduced capsaicin-induced calcitonin gene-related peptide (CGRP) release. Coadministration of SR144528 resulted in a rightforward shift (pKB 8.1 and 8.2 for (+)-AM1241 and L768242, respectively) of the dose-response curve. Experiments on capsaicin-induced CGRP release in tissue from CB1-/- mice ruled out a CB1-mediated effect. These results confirm that CB2 is present in the central nervous system and suggest that CB2 agonists may elicit their analgesic effect by acting not only at non-neuronal peripheral sites but also at neural level, making CB2 an attractive target for chronic pain treatment.

Analgesics↗
PubMed2003

Fractalkine protein localization and gene expression in mouse brain.

Few chemokines are expressed constitutively in the brain at detectable levels; amongst them is fractalkine. We analyzed the distribution of fractalkine in the mouse brain with the aim of giving a neuroanatomical support to the study of its physiological function. To this end, we carried out an analysis of fractalkine protein localization and gene expression. An anti-fractalkine antibody was produced and used to perform an immunohistochemical study. The results indicated a high level of fractalkine protein in cortex, hippocampus, basal ganglia, and olfactory bulb. In particular, the presence of abundant immunoreactive neurons was observed in layers II, III, V, and VI of the cortex. In the hippocampus, the CA1 region was the most intensely labeled, but immunoreactive neurons were present also in CA2 and CA3, whereas in the basal ganglia, immunoreactive cells were observed in the caudate putamen. Other brain structures such as the brainstem showed a few scattered immunoreactive cells. The presence of fractalkine immunoreactive fibers was revealed only in the olfactory bulb and in the anterior olfactory nuclei. Gene expression study results, obtained by both semiquantitative PCR and in situ hybridization, matched protein localization with the highest levels of fractalkine transcript detected in the hippocampus, cortex, and striatum. The present study showed that fractalkine protein and mRNA are constitutively expressed at a high level in forebrain structure, but are almost absent in the hindbrain. Furthermore, localization at the cellular body level would suggest a paracrine or cell-to-cell interaction role for fractalkine more than a neurotransmission modulatory function.

Amygdala↗
PubMed2002

Relationships between CB1 cannabinoid receptors and pituitary endocrine cells in Xenopus laevis: an immunohistochemical study.

The distribution of the cannabinoid CB1 receptor and its relationships with individual endocrine cell types were investigated by immunohistochemistry in the anterior lobe of the Xenopus adenohypophysis. By use of a specific primary antibody raised in rabbit against the amino terminus of the rat CB1, we have found numerous CB1-like-immunoreactive cells distributed throughout all of the pituitary anterior lobe with the exception of the ventrocranial area adjacent to the median eminence of the neurohypophysis. Aided by both double-immunostaining on consecutive serial sections and double-simultaneous immunofluorescence on the same section of the gland, the CB1-like immunoreactivity was compared to some specific hormone immunoreactive cells. CB1 labelings were mainly codistributed, and even colocalized, with lactotrophs and thyrotrophs. Gonadotrophs containing CB1 receptors were also observed. In contrast, corticotrophs, which are located mainly in the ventrocranial pole of the anterior lobe, were generally devoid of CB1. Since nerve terminals immunoreactive to the CB1 antibody were observed within the vascular zone of the median eminence, the possibility that endocannabinoids are involved in the control of some secretory activities of Xenopus pituitary, either indirectly via hypothalamic neurosecretory mechanisms or directly on the pituitary cells, was envisaged. In particular, the present study suggests the occurrence of a direct cannabinergic modulation of the prolactin, gonadotrophin, and thyrotrophin secretions through the CB1 receptor.

Adrenocorticotropic Hormone↗
PubMed2001

Cannabinoid receptor CB1-like and glutamic acid decarboxylase-like immunoreactivities in the brain of Xenopus laevis.

Investigation of the cannabinoid system in a vertebrate group phylogenetically distant from mammals might improve understanding of its physiological role. Thus, in the present study, the distribution of the cannabinoid CB1 receptor has been investigated in the brain of Xenopus laevis (anuran amphibians) by immunohistochemistry, using both light and confocal laser-scanning microscopy. Immunostained neuronal perikarya and terminals were found in the olfactory bulb, dorsal and medial pallium, striatum, and amygdala. Varicosities and nerve terminals containing CB1-like immunoreactivity were also seen in the thalamus and hypothalamus. A number of stained cells were observed in the pars distalis of the pituitary gland. Positive nerve fibers were distributed throughout mesencephalic tegmentum, and in the cerebellum immunolabeling was observed in some Purkinje and possibly Golgi cells. The confocal microscopic analysis of CB1-like and glutamic acid decarboxylase-like immunoreactivities in both the medial pallium of the telencephalon and the olfactory bulbs showed a wide codistribution of the two markers. The present results indicate that distribution of CB1 is conserved in the course of phylogeny. Furthermore, the close relationship between CB1-like and glutamic acid decarboxylase-like immunolabelings point toward the existence of a functional link between cannabinergic and GABAergic innervations also in amphibian brain.

Animals↗
PubMed2001

Mechanisms of endocannabinoid inactivation: biochemistry and pharmacology.

The endocannabinoids, a family of endogenous lipids that activate cannabinoid receptors, are released from cells in a stimulus-dependent manner by cleavage of membrane lipid precursors. After release, the endocannabinoids are rapidly deactivated by uptake into cells and enzymatic hydrolysis. Endocannabinoid reuptake occurs via a carrier-mediated mechanism, which has not yet been molecularly characterized. Endocannabinoid reuptake has been demonstrated in discrete brain regions and in various tissues and cells throughout the body. Inhibitors of endocannabinoid reuptake include N-(4-hydroxyphenyl)-arachidonylamide (AM404), which blocks transport with IC50 (concentration necessary to produce half-maximal inhibition) values in the low micromolar range. AM404 does not directly activate cannabinoid receptors or display cannabimimetic activity in vivo. Nevertheless, AM404 increases circulating anandamide levels and inhibits motor activity, an effect that is prevented by the CB1 cannabinoid antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A). AM404 also reduces behavioral responses to dopamine agonists and normalizes motor activity in a rat model of attention deficit hyperactivity disorder. The endocannabinoids are hydrolyzed by an intracellular membrane-bound enzyme, termed anandamide amidohydrolase (AAH), which has been molecularly cloned. Several fatty acid sulfonyl fluorides inhibit AAH activity irreversibly with IC50 values in the low nanomolar range and protect anandamide from deactivation in vivo. alpha-Keto-oxazolopyridines inhibit AAH activity with high potency (IC50 values in the low picomolar range). A more thorough characterization of the roles of endocannabinoids in health and disease will be necessary to define the significance of endocannabinoid inactivation mechanisms as targets for therapeutic drugs.

Amidohydrolases↗
PubMed2000

Inhibition of caspase-1-like activity by Ac-Tyr-Val-Ala-Asp-chloromethyl ketone induces long-lasting neuroprotection in cerebral ischemia through apoptosis reduction and decrease of proinflammatory cytokines.

Broad spectrum caspase inhibitors have been found to reduce neurodegeneration caused by cerebral ischemia. We studied whether blockade of group I caspases, mainly caspase-1, using the inhibitor Ac-YVAD.cmk reduced infarct volume and produced prolonged neuroprotection. Ac-YVAD.cmk (300 ng/rat) was injected intracerebroventricularly 10 min after permanent middle cerebral artery occlusion in the rat. Drug treatment induced a significant reduction of infarct volume not only 24 hr after ischemia (total damage, percentage of hemisphere volume: control, 41.1 +/- 2.3%; treated, 26.5 +/- 2.1%; p < 0.05) but also 6 d later (total damage: control, 30.6 +/- 2.2%; treated, 23.0 +/- 2.2%; p < 0.05). Ac-YVAD. cmk treatment resulted in a reduction not only of caspase-1 (control, 100 +/- 20.3%; treated, 3.4 +/- 10.4%; p < 0.01) but also of caspase-3 (control, 100 +/- 30.3%; treated, 13.2 +/- 9.5%; p < 0.05) activity at 24 hr and led to a parallel decrease of apoptosis as measured by nucleosome quantitation (control, 100 +/- 11.8%; treated, 47 +/- 5.9%; p < 0.05). Six days after treatment no differences in these parameters could be detected between control and treated animals. Likewise, brain levels of the proinflammatory cytokines IL-1beta and TNF-alpha were reduced at 24 hr (39.5 +/- 23.7 and 51.9 +/- 10.3% of control, respectively) but not at 6 d. Other cytokines, IL-10, MCP-1, MIP-2, and the gaseous mediator nitric oxide, were not modified by the treatment. These findings indicate that blockade of caspase-1-like activity induces a long-lasting neuroprotective effect that, in our experimental conditions, takes place in the early stages of damage progression. Finally, this effect is achieved by interfering with both apoptotic and inflammatory mechanisms.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Reversal of dopamine D(2) receptor responses by an anandamide transport inhibitor.

We characterized the pharmacological properties of the anandamide transport inhibitor N-(4-hydroxyphenyl)-arachidonamide (AM404) in rats and investigated the effects of this drug on behavioral responses associated with activation of dopamine D(2) family receptors. Rat brain slices accumulated [(3)H]anandamide via a high-affinity transport mechanism that was blocked by AM404. When administered alone in vivo, AM404 caused a mild and slow-developing hypokinesia that was significant 60 min after intracerebroventricular injection of the drug and was reversed by the CB1 cannabinoid receptor antagonist SR141716A. AM404 produced no significant catalepsy or analgesia, two typical effects of direct-acting cannabinoid agonists. However, AM404 prevented the stereotypic yawning produced by systemic administration of a low dose of apomorphine, an effect that was dose-dependent and blocked by SR141716A. Furthermore, AM404 reduced the stimulation of motor behaviors elicited by the selective D(2) family receptor agonist quinpirole. Finally, AM404 reduced hyperactivity in juvenile spontaneously hypertensive rats, a putative model of attention deficit hyperactivity disorder. The results support a primary role of the endocannabinoid system in the regulation of psychomotor activity and point to anandamide transport as a potential target for neuropsychiatric medicines.

Animals↗
PubMed2000

Carrier-mediated transport and enzymatic hydrolysis of the endogenous cannabinoid 2-arachidonylglycerol.

The human astrocytoma cell line CCF-STTGI accumulates [3H]2-AG through an Na(+)- and energy-independent process, with a Km of 0.7 +/- 0.1 microM. Non-radioactive 2-AG, anandamide or the anandamide transport inhibitor 4-hydroxyphenyl arachidonamide inhibit [3H]2-AG uptake with half-maximal inhibitory concentrations (IC50) of 5.5 +/- 1.0 microM, 4.2 +/- 0.3 microM and 1.8 = 0.1 microM, respectively. A variety of lipid transport substrates and inhibitors interfere with neither [3H]2-AG nor [3H]anandamide uptake. These results suggest that 2-AG and anandamide are internalized in astrocytoma cells through a common carrier-mediated mechanism. After incubation with [3H]2-AG, radioactivity is recovered in phospholipids, monoacylglycerols (unmetabolized [3H]2-AG), free fatty acids ([3H]arachidonate) and, to a minor extent, diacylglycerols and triacylglycerols. Arachidonic acid (100 microM) and triacsin C (10 microM), an acyl-CoA synthetase inhibitor, prevent incorporation of [3H]arachidonic acid in phospholipids and significantly reduce [3H]2-AG transport. Thus, the driving force for 2-AG internalization may derive from the hydrolysis of 2-AG to arachidonate and the subsequent incorporation of this fatty acid into phospholipids.

Arachidonic Acid↗
PubMed1999

Structural determinants for recognition and translocation by the anandamide transporter.

The biological actions of anandamide (arachidonylethanolamide), an endogenous cannabinoid lipid, are terminated by a two-step inactivation process consisting of carrier-mediated uptake and intracellular hydrolysis. Anandamide uptake in neurons and astrocytes is mediated by a high-affinity, Na+-independent transporter that is selectively inhibited by N-(4-hydroxyphenyl)-arachidonamide (AM404). In the present study, we examined the structural determinants governing recognition and translocation of substrates by the anandamide transporter constitutively expressed in a human astrocytoma cell line. Competition experiments with a select group of analogs suggest that substrate recognition by the transporter is favored by a polar nonionizable head group of defined stereochemical configuration containing a hydroxyl moiety at its distal end. The secondary carboxamide group interacts favorably with the transporter, but may be replaced with either a tertiary amide or an ester, suggesting that it may serve as hydrogen acceptor. Thus, 2-arachidonylglycerol, a putative endogenous cannabinoid ester, also may serve as a substrate for the transporter. Substrate recognition requires the presence of at least one cis double bond situated at the middle of the fatty acid carbon chain, indicating a preference for ligands whose hydrophobic tail can adopt a bent U-shaped conformation. On the other hand, uptake experiments with radioactively labeled substrates show that no fewer than four cis nonconjugated double bonds are required for optimal translocation across the cell membrane, suggesting that substrates are transported in a folded hairpin conformation. These results outline the general structural requisites for anandamide transport and may assist in the development of selective inhibitors with potential clinical applications.

Arachidonic Acids↗
PubMed1999

Anandamide transport inhibition by the vanilloid agonist olvanil.

The structural similarities between the anandamide transport inhibitor N-(4-hydroxyphenyl)-arachidonylamide (AM404) and the synthetic vanilloid agonist olvanil [(N-vanillyl)-9-oleamide], prompted us to investigate the possibility that olvanil may interfere with anandamide transport. The intracellular accumulation of [3H]anandamide by human astrocytoma cells was prevented by olvanil with a Ki value of 14.1+/-7.1 microM. By contrast, capsaicin [(8-methyl-N-vanillyl)-6-noneamide], a plant-derived vanilloid agonist, and capsazepine (N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2 H-2-benzazepine-2-carbothioamide), a vanilloid antagonist, had no such effect (Ki > 100 microM). These results indicate that, although less potent than AM404 (Ki 2.1+/-0.2 microM), olvanil may reduce anandamide clearance at concentrations similar to those needed for vanilloid receptor activation.

Amidohydrolases↗
PubMed1998

Immunolocalization of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, dopamine, and serotonin in the forebrain of Ambystoma mexicanum.

To improve basic knowledge about the neurochemical organization of the urodele brain, and to study discrepancies in the localization of monoaminergic markers, we immunohistochemically charted the distribution of four such markers (tyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine, and serotonin) in the axolotl (Ambystoma mexicanum) forebrain. Catecholaminergic and serotoninergic systems were found in similar locations to those seen in other Urodela. As seen in other vertebrates, the localization of the different monoaminergic markers reveals some inconsistencies. Cells that are exclusively tyrosine hydroxylase-immunoreactive are observed in the olfactory bulb, anterior olfactory nucleus/nucleus accumbens region, the epichiasmatic portion of the preoptic nucleus, and in the pars intercalaris thalami, whereas cells that are only labelled by aromatic L-amino acid decarboxylase are seen in the anterior olfactory nucleus/nucleus accumbens region, the bed nuclei of the anterior commissure, the posterior portion of the preoptic nucleus, the ventral hypothalamus, and the pars intercalaris thalami. The presence of cells solely serotonin (5-HT)-immunoreactive is suggested for the nucleus infundibularis dorsalis. Conversely, there were no areas that appeared to be exclusively immunoreactive for dopamine. Double-labelling for aromatic L-amino acid decarboxylase/tyrosine hydroxylase and aromatic L-amino acid decarboxylase/serotonin, together with cell counting, confirmed the existence of neurons that express only one monoaminergic marker in amphibian, supporting the hypothesis that these cells are universally present in the central nervous system of vertebrates.

Ambystoma↗
PubMed1997

Inhibition of intestinal motility by anandamide, an endogenous cannabinoid.

The endogenous cannabinoid ligand anandamide (arachidonylethanolamide) inhibited the intestinal passage of a charcoal meal when administered s.c. in mice at doses ranging from 0.1 to 50 mg/kg. This effect was prevented by the cannabinoid CB1 receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide x HCl] (1 mg/kg s.c.), but it was not affected by the anandamide transport inhibitor, N-(4-hydroxyphenyl) arachidonylethanolamide (AM404) (50 mg/kg, s.c.). The results indicate that anandamide modulates intestinal motility in mice by activating cannabinoid CB1 receptors. They also suggest that anandamide transport, which was previously shown to participate in terminating neural and vascular responses to anandamide, does not contribute to anandamide inactivation in intestinal tissue.

Animals↗
PubMed1997

Potentiation of anandamide hypotension by the transport inhibitor, AM404.

The putative endogenous cannabinoid, anandamide (0.2-2 mg/kg i.v.), decreased systemic blood pressure dose-dependently in anesthesized guinea pigs. These effects were prevented by the CB1 cannabinoid receptor antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide x HCl] at the dose of 0.2 mg/kg i.v. The vasodepressor responses to anandamide were significantly potentiated and prolonged by a novel inhibitor of carrier-mediated anandamide transport, N-(4-hydroxyphenyl) arachidonylethanolamide (AM404) (10 mg/kg, i.v.). These results suggest that anandamide transport participates in terminating the vascular actions of anandamide.

Animals↗
PubMed1997

Functional role of high-affinity anandamide transport, as revealed by selective inhibition.

Anandamide, an endogenous ligand for central cannabinoid receptors, is released from neurons on depolarization and rapidly inactivated. Anandamide inactivation is not completely understood, but it may occur by transport into cells or by enzymatic hydrolysis. The compound N-(4-hydroxyphenyl)arachidonylamide (AM404) was shown to inhibit high-affinity anandamide accumulation in rat neurons and astrocytes in vitro, an indication that this accumulation resulted from carrier-mediated transport. Although AM404 did not activate cannabinoid receptors or inhibit anandamide hydrolysis, it enhanced receptor-mediated anandamide responses in vitro and in vivo. The data indicate that carrier-mediated transport may be essential for termination of the biological effects of anandamide, and may represent a potential drug target.

Analgesics↗
PubMed1997

Inhibition of anandamide hydrolysis in rat brain tissue by (E)-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one.

Anandamide, an endogenous canabinoid substance, is hydrolyzed by an amidohydrolase activity present in rat brain and liver. We report that the bromoenol lactone, (E)-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BTNP), is a potent inhibitor of this enzyme activity. BTNP prevented anandamide hydrolysis in rat brain microsomes with an IC50 of 0.8 +/- 0.3 microM. Kinetic and dialysis experiments indicated that this effect was non-competitive and irreversible. After chromatographic fractionation of the enzyme activity, BTNP was still effective, suggesting that it interacts directly with the enzyme. Anandamide hydrolysis was 12-fold greater in rat cortical neurons (1.94 +/- 0.1 pmol/min/mg protein) than in cortical astrocytes (0.16 +/- 0.01 pmol/min/mg protein) and, in either cell type, it was inhibited by BTNP (IC50 = 0.1 microM in neurons). These results suggest that BTNP may provide a useful lead for the development of novel inhibitors of anandamide hydrolysis.

Amidohydrolases↗
PubMed1997

Long-lasting effect of catecholamine deficiency on differentiating vasopressin and oxytocin neurons in the rat supraoptic nucleus.

According to our earlier study, the catecholamine depletion in neonatal rats resulted in stimulation of the vasopressin and oxytocin gene expression in the neurons of the supraoptic nucleus. The present study extends this line, evaluating whether the catecholamine deficiency provides a long-lasting effect on the differentiating vasopressin and oxytocin neurons of the supraoptic nucleus. Catecholamines were depleted by daily injections of an inhibitor of the catecholamine synthesis, alpha-methyl-p-tyrosine, first, to pregnant rats from the 9th to the 21st day of gestation and, then, to their pups from the 2nd to the 10th postnatal day. The animals, injected with saline instead of drugs, served as controls. The pharmacologically-treated and control rats were kept for four months under normal laboratory conditions until processing the materials for semi-quantitative in situ hybridization and immunocytochemistry of vasopressin and oxytocin messenger RNAs and peptides, respectively. There were no differences in the vasopressin and oxytocin messenger RNA concentrations in the supraoptic nucleus in rats following preliminary catecholamine depletion compared to controls. Conversely, the catecholamine deficiency resulted in an increased content of the vasopressin-immunoreactive material in cell bodies and processes. This was also the case for the oxytocin-immunoreactive cell bodies but only in females, suggesting an interference of catecholamines with sexual steroids in their action. The number and size of vasopressin and oxytocin neurons did not change in pharmacologically-treated rats compared to the controls. Thus, the catecholamine deficiency in the course of the neuron differentiation resulted in a long-lasting augmentation of the intracellular content of vasopressin and oxytocin but did not influence the vasopressin and oxytocin gene expression. This might be explained rather by the reduced level of peptide release than by an increased level of the peptide production.

Animals↗