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Mauro Maccarrone

Publications and source records attributed to Mauro Maccarrone.

At least 19 recordsLinked to original sources
PubMed2006

Closing the gate to the active site: effect of the inhibitor methoxyarachidonyl fluorophosphonate on the conformation and membrane binding of fatty acid amide hydrolase.

Fatty acid amide hydrolase (FAAH) is a dimeric, membranebound enzyme that degrades neuromodulatory fatty acid amides and esters and is expressed in mammalian brain and peripheral tissues. The cleavage of approximately 30 amino acids from each subunit creates an FAAH variant that is soluble and homogeneous in detergent-containing buffers, opening the avenue to the in vitro mechanistic and structural studies. Here we have studied the stability of FAAH as a function of guanidinium hydrochloride concentration and of hydrostatic pressure. The unfolding transition was observed to be complex and required a fitting procedure based on a three-state process with a monomeric intermediate. The first transition was characterized by dimer dissociation, with a free energy change of approximately 11 kcal/mol that accounted for approximately 80% of the total stabilization energy. This process was also paralleled by a large change in the solvent-accessible surface area, because of the hydration occurring both at the dimeric interface and within the monomers. As a consequence, the isolated subunits were found to be much less stable (DeltaG approximately 3 kcal/mol). The addition of methoxyarachidonyl fluorophosphonate, an irreversible inhibitor of FAAH activity, enhanced the stability of the dimer by approximately 2 kcal/mol, toward denaturant- and pressure-induced unfolding. FAAH inhibition by methoxyarachidonyl fluorophosphonate also reduced the ability of the protein to bind to the membranes. These findings suggest that local conformational changes at the level of the active site might induce a tighter interaction between the subunits of FAAH, affecting the enzymatic activity and the interaction with membranes.

Amidohydrolases↗
PubMed2006

Follicle-stimulating hormone activates fatty acid amide hydrolase by protein kinase A and aromatase-dependent pathways in mouse primary Sertoli cells.

Among the biological activities of the endocannabinoid anandamide (N-arachidonoylethanolamine) (AEA), growing interest has been attracted by the regulation of mammalian fertility. Recently we have shown that treatment of mouse primary Sertoli cells with FSH enhances the activity of the AEA hydrolase [fatty acid amide hydrolase (FAAH)], though the molecular details were not elucidated. Here, we investigated whether FSH was also able to affect the enzymes that synthesize AEA (N-acyltransferase and N-acyl-phosphatidyl-ethanolamine-phospholipase D), the endogenous content of this endocannabinoid, and the level of the AEA-binding vanilloid receptor 1 (transient receptor potential channel vanilloid receptor subunit 1). We show that FSH enhanced FAAH activity (up to approximately 500% of the controls) and expression (up to approximately 300%), leading to a marked reduction (down to approximately 15%) of AEA content. However N-acyltransferase and N-acyl-phosphatidyl-ethanolamine-phospholipase D activity, and transient receptor potential channel vanilloid receptor subunit 1 binding were not affected. We also show that diacylglycerol lipase and monoacylglycerol lipase, which respectively synthesize and degrade 2-arachidonoyl-glycerol, were not regulated by FSH, neither was the membrane transport of this endocannabinoid. In addition, we show that FAAH stimulation by FSH was abrogated by inhibitors of protein kinase A (PKA) and cytochrome-P(450) aromatase, and was conversely mimicked by N,O'-dibutyryl cAMP and estrogen. Finally, we demonstrate that FSH protects Sertoli cells against the pro-apoptotic activity of AEA, through PKA and aromatase-dependent activation of FAAH. Altogether these data suggest that FAAH is the only target of FSH among the elements of the endocannabinoid system, and that its regulation by PKA and aromatase-dependent pathways impacts Sertoli cell proliferation.

Amidohydrolases↗
PubMed2006

Effect of lipid rafts on Cb2 receptor signaling and 2-arachidonoyl-glycerol metabolism in human immune cells.

Recently, we have shown that treatment of rat C6 glioma cells with the raft disruptor methyl-beta-cyclodextrin (MCD) doubles the binding of anandamide (AEA) to type-1 cannabinoid receptors (CB1R), followed by CB1R-dependent signaling via adenylate cyclase and p42/p44 MAPK activity. In the present study, we investigated whether type-2 cannabinoid receptors (CB2R), widely expressed in immune cells, also are modulated by MCD. We show that treatment of human DAUDI leukemia cells with MCD does not affect AEA binding to CB2R, and that receptor activation triggers similar [35S]guanosine-5'-O-(3-thiotriphosphate) binding in MCD-treated and control cells, similar adenylate cyclase and MAPK activity, and similar MAPK-dependent protection against apoptosis. The other AEA-binding receptor transient receptor potential channel vanilloid receptor subunit 1, the AEA synthetase N-acyl-phosphatidylethanolamine-phospholipase D, and the AEA hydrolase fatty acid amide hydrolase were not affected by MCD, whereas the AEA membrane transporter was inhibited (approximately 55%) compared with controls. Furthermore, neither diacylglycerol lipase nor monoacylglycerol lipase, which respectively synthesize and degrade 2-arachidonoylglycerol, were affected by MCD in DAUDI or C6 cells, whereas the transport of 2-arachidonoylglycerol was reduced to approximately 50%. Instead, membrane cholesterol enrichment almost doubled the uptake of AEA and 2-arachidonoylglycerol in both cell types. Finally, transfection experiments with human U937 immune cells, and the use of primary cells expressing CB1R or CB2R, ruled out that the cellular environment could account per se for the different modulation of CB receptor subtypes by MCD. In conclusion, the present data demonstrate that lipid rafts control CB1R, but not CB2R, and endocannabinoid transport in immune and neuronal cells.

Arachidonic Acids↗
PubMed2006

N-arachidonoyl-dopamine tunes synaptic transmission onto dopaminergic neurons by activating both cannabinoid and vanilloid receptors.

In the present study, we used electrophysiological, biochemical, and confocal microscopy techniques, to investigate the functional role of transient receptor potential vanilloid type 1 (TRPV1) and cannabinoid type 1 receptors (CB1-R) in the substantia nigra pars compacta (SNpc) and their stimulation by the endocannabinoid N-arachidonoyl-dopamine (NADA). Liquid chromatography-mass spectrometry analyses revealed that a NADA-like compound is produced in substantia nigra slices, in conditions of hyperactivity. Moreover, the functional role of both TRPV1 and CB1-R in modulating synaptic transmission in this area was suggested by confocal microscopy data, showing TRPV1 and CB1-R immunoreactivity in punctate structures, probably representing synaptic contacts on cell bodies of the SNpc. In patch-clamp recordings from dopamine (DA) neurons of the SNpc, we found that NADA increases or reduces glutamatergic transmission onto DA neurons by activating TRPV1 and CB1 receptors, respectively, whereas it decreases GABAergic transmission via CB1 stimulation. Facilitation of glutamate release through TRPV1 was blocked in the presence of a selective blocker of the putative endocannabinoid membrane transporter (EMT), indicating that NADA needs to be taken up by cells to interact with this receptor. In line with these data, biochemical results demonstrated that NADA selectively acted at CB1-R when its re-uptake was blocked. Altogether these data demonstrate a significant role exerted by the endocannabinoid/endovanilloid NADA in the regulation of synaptic transmission to DA neurons of the SNpc. Moreover, they highlight a key function of the EMT transporter in promoting the stimulation of TRPV1 or CB1-R, thus favoring facilitation or inhibition of glutamate synaptic release.

Animals↗
PubMed2006

Jekyll and hyde: two faces of cannabinoid signaling in male and female fertility.

Mammalian reproduction is a complicated process designed to diversify and strengthen the genetic complement of the offspring and to safeguard regulatory systems at various steps for propagating procreation. An emerging concept in mammalian reproduction is the role of endocannabinoids, a group of endogenously produced lipid mediators, that bind to and activate cannabinoid receptors. Although adverse effects of cannabinoids on fertility have been implicated for years, the mechanisms by which they exert these effects were not clearly understood. With the identification of cannabinoid receptors, endocannabinoid ligands, their key synthetic and hydrolytic pathways, and the generation of mouse models missing cannabinoid receptors, a wealth of information on the significance of cannabinoid/endocannabinoid signaling in spermatogenesis, fertilization, preimplantation embryo development, implantation, and postimplantation embryonic growth has been generated. This review focuses on various aspects of the endocannabinoid system in male and female fertility. It is hoped that a deeper insight would lead to potential clinical applications of the endocannabinoid signaling as a target for correcting infertility and improving reproductive health in humans.

Cannabinoid Receptor Modulators↗
PubMed2006

Stage-variations of anandamide hydrolase activity in the mouse uterus during the natural oestrus cycle.

Recent studies have demonstrated that the endogenous cannabinoids are important modulators of fertility in mammals. In particular, a role of the endocannabinoid system in early stages of embryo development, oviductal transport of embryos, pregnancy maintenance and labour has been demonstrated in rodents and/or in humans. In the present paper, we report the analysis of FAAH activity and protein content in the mouse uterus as a function of the natural oestrus cycle stages. Variations of FAAH activity are discussed in relationship to changes in sex steroid levels and to the possible action of AEA on remodelling of uterine tissues.

Journal Article↗
PubMed2006

Methods to assay anandamide hydrolysis and transport in synaptosomes.

Anandamide (AEA) is the most studied member of a new class of lipid mediators, collectively called endocannabinoids. The biological activity of AEA at cannabinoid and noncannabinoid receptors depends on its life span in the extracellular space, which is regulated by a rapid cellular uptake, followed by intracellular degradation by the enzyme AEA hydrolase (fatty acid amide hydrolase). Here, we present the methodological details of the procedures that we have developed to assay fatty acid amide hydrolase activity and to characterize AEA transport through cell membranes in a new ideal ex vivo system like brain synaptosomes.

Animals↗
PubMed2006

Fatty acid amide hydrolase: a potential target for next generation therapeutics.

Endocannabinoids are amides, esters and ethers of long chain polyunsaturated fatty acids, which act as new lipid mediators. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol are the main endogenous agonists of cannabinoid receptors, able to mimic several pharmacological effects of Delta(9)-tetrahydrocannabinol, the active principle of Cannabis sativa preparations like hashish and marijuana. The activity of AEA at its receptors is limited by cellular uptake through a specific membrane transporter, followed by intracellular degradation by a fatty acid amide hydrolase (FAAH). Growing evidence demonstrates that FAAH is the critical regulator of the endogenous levels of AEA, suggesting that it may serve as an attractive therapeutic target for the treatment of human disorders. In particular, FAAH inhibitors may be next generation therapeutic drugs of potential value for the treatment of pathologies in the central nervous system and in the periphery. Here, the potential applications of these inhibitors for human disease will be reviewed, with an emphasis on the properties of hydro(pero)xy-anandamides. In fact, these oxygenated derivatives of AEA are the most powerful inhibitors of FAAH of natural origin as yet discovered. In addition, new insights into the promoter region of FAAH gene will be presented, and the therapeutic potential of mimetics of transcription factors of this gene in the management of human infertility will be discussed.

Amidohydrolases↗
PubMed2006

Regulation by cannabinoid receptors of anandamide transport across the blood-brain barrier and through other endothelial cells.

The endocannabinoid anandamide (AEA) has many neurovascular activities. However, it is not yet clear how AEA can be metabolized at the neurovascular interface, and how it can move through the vascular and the cerebral compartments. The results reported in this article show that isolated bovine brain microvessels, an ex vivo model of the blood-brain barrier, have detectable levels of endogenous AEA and possess the biochemical machinery to bind and metabolize it, i.e. type-1 and type-2 cannabinoid receptors (CB1R and CB2R), a selective AEA membrane transporter (AMT), an AEA-degrading fatty acid amide hydrolase, and the AEA-synthesizing enzymes N-acyltransferase and N-acyl-phosphatidylethanolamines-specific phospholipase D. We also show that activation of CB1R enhances AMT activity through increased nitric oxide synthase (NOS) activity and subsequent increase of NO production. AMT activity is instead reduced by activation of CB2R, which inhibits NOS and NO release. In addition, binding experiments and immunoelectronmicroscopy demonstrate that different endothelial cells vary in the expression of CB1R and CB2R on the luminal and/or abluminal sides. The different localization of CBRs can lead to a diverse effect on AMT activity on the luminal and abluminal membranes, suggesting that the distribution of these receptors may drive AEA directional transport through the blood-brain barrier and other endothelial cells.

Amidohydrolases↗
PubMed2005

Further insights into the regulation of human FAAH by progesterone and leptin implications for endogenous levels of anandamide and apoptosis of immune and neuronal cells.

We have recently reported that leptin (L) and progesterone (P) stimulate the activity and the expression of the endocannabinoid-degrading enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH) in human lymphoma U937 cells, but not in human neuroblastoma CHP100 cells. We have also shown that leptin and progesterone do not affect the proteins of the endocannabinoid system that synthesize and transport AEA. Here, we have summarized these findings, and have extended them by investigating the effect of leptin and progesterone on the endogenous levels of AEA. We show that leptin and progesterone significantly reduce AEA content in U937 cells (down to approximately 20% and approximately 50% of the controls, respectively), whereas they are ineffective on AEA levels in CHP100 cells. In addition, we show that leptin and progesterone prevent the pro-apoptotic activity of AEA in U937 cells, reducing DNA fragmentation by approximately 50% and approximately 35% compared to controls, respectively. Instead, neither hormone affects apoptosis induced by AEA in CHP100 cells. Since the anti-apoptotic activity of leptin and progesterone parallels their effect on FAAH, it can be suggested that enhanced degradation of AEA is the means to protect U937 cells against the toxicity of this compound. Altogether, these data suggest that a cell-specific regulation of FAAH gene might modulate the apoptotic potential of endocannabinoids along the neuroimmune axis. These findings might be relevant for the development of cell-selective drugs targeted towards FAAH.

Amidohydrolases↗
PubMed2005

Characterization of the endocannabinoid system in boar spermatozoa and implications for sperm capacitation and acrosome reaction.

Anandamide (AEA) is the endogenous ligand of cannabinoid (CB) receptors, and as such it plays several central and peripheral activities. Regulation of female fertility by AEA has attracted growing interest, yet a role for this endocannabinoid in controlling sperm function and male fertility in mammals has been scarcely investigated. In this study we report unprecedented evidence that boar sperm cells have the biochemical machinery to bind and degrade AEA, i.e. type-1 cannabinoid receptors (CB1R), vanilloid receptors (TRPV1), AEA-synthesizing phospholipase D (NAPE-PLD), AEA transporter (AMT) and AEA hydrolase (FAAH). We also show that the non-hydrolyzable AEA analogue methanandamide reduces sperm capacitation and, as a consequence, inhibits the process of acrosome reaction (AR) triggered by the zona pellucida, according to a cyclic AMP-dependent pathway triggered by CB1R activation. Furthermore, activation of TRPV1 receptors seems to play a role of stabilization of the plasma membranes in capacitated sperm, as demonstrated by the high incidence of spontaneous AR occurring during the cultural period when TRPV1 activity was antagonized by capsazepine. We show that sperm cells have a complete and efficient endocannabinoid system, and that activation of cannabinoid or vanilloid receptors controls, at different time-points, sperm functions required for fertilization. These observations open new perspectives on the understanding and treatment of male fertility problems.

Acrosome Reaction↗
PubMed2005

Central and peripheral interactions between endocannabinoids and steroids, and implications for drug dependence.

Endocannabinoids are biologically active amides, esters and ether of long chain polyunsaturated fatty acids. They interact with several neurotransmitters in the central nervous system (CNS), and with various signaling molecules (including cytokines) in the periphery. Critical interactions have emerged also with steroids, another group of well-known bioactive lipids, both centrally and peripherally. Here, I briefly review the targets of the combined action of endocannabinoids and steroids, and the available evidence concerning the direct regulation by the latter compounds of the proteins of the endocannabinoid system (ES). In addition, I discuss recent examples of endocannabinoids and steroids working together in the central nervous system and in the periphery, which allowed to disclose some molecular details of the interactions between these two groups of lipids. Taken together, available data suggest that steroids can modulate the endocannabinoid tone, through genomic or nongenomic regulation, and that endocannabinoids can complement the biological activity of steroids. In this line, the issues concerning the tissue- and species-specificity of the endocannabinoid-steroid interface, and the possibility that also endocannabinoids may modulate steroid metabolism, are addressed. Finally, I present the hypothesis that retrograde endocannabinoid signaling, by reducing striatal glutamate release, may be part of the molecular events responsible for the influence of steroids on drug abuse.

Blood Cells↗
PubMed2005

Cholesterol-dependent modulation of type 1 cannabinoid receptors in nerve cells.

Type 1 cannabinoid receptors (CB1R) are G-protein-coupled receptors that mediate several actions of the endocannabinoid anandamide (N-arachidonoylethanolamine; AEA) in the central nervous system. Here we show that cholesterol enrichment of rat C6 glioma cell membranes reduces by approximately twofold the binding efficiency (i.e., the ratio between maximum binding and dissociation constant) of CB1R and that activation of CB1R by AEA leads to approximately twofold lower [(35)S]GTPgammaS binding in cholesterol-treated cells than in controls. In addition, we show that CB1R-dependent signaling via adenylate cyclase and p42/p44 mitogen-activated protein kinase is almost halved by cholesterol enrichment. Unlike CB1R, the other AEA-binding receptor TRPV1, the AEA synthetase NAPE-PLD, and the AEA hydrolase FAAH are not modulated by cholesterol, whereas the catalytic efficiency (i.e., the ratio between maximal velocity and Michaelis-Menten constant) of the AEA membrane transporter AMT is almost doubled compared with control cells. These data demonstrate that, among the proteins of the "endocannabinoid system," only CB1R and AMT critically depend on membrane cholesterol content. This observation may have important implications for the role of CB1R in protecting nerve cells against (endo)cannabinoid-induced apoptosis.

Animals↗
PubMed2005

Abnormal sensitivity to cannabinoid receptor stimulation might contribute to altered gamma-aminobutyric acid transmission in the striatum of R6/2 Huntington's disease mice.

BACKGROUND: One of the earliest neurochemical alterations observed in both Huntington's disease (HD) patients and HD animal models is the dysregulation of the endocannabinoid system, an alteration that precedes the development of identifiable striatal neuropathology. How this alteration impacts striatal synaptic transmission is unknown. METHODS: We measured the effects of cannabinoid receptor stimulation on gamma-aminobutyric acid (GABA)-ergic synaptic currents recorded from striatal neurons of R6/2 HD mice in the early phase of their disease. RESULTS: The sensitivity of striatal GABA synapses to cannabinoid receptor stimulation is severely impaired in R6/2 HD mice. In particular, whereas in control animals activation of cannabinoid CB1 receptors results in a significant inhibition of both evoked and spontaneous GABA-mediated synaptic events by a presynaptic mechanism, in R6/2 mice this treatment fails to reduce GABA currents but causes, in contrast, a slight increase of spontaneous inhibitory postsynaptic currents (sIPSCs). CONCLUSIONS: Experimental HD was also associated with enhanced frequency of sIPSCs, a result consistent with the conclusion that loss of cannabinoid-mediated control of GABA transmission might contribute to hyperactivity of GABA synapses in the striatum of HD mice. Accordingly, spontaneous excitatory postsynaptic currents, which were not upregulated in R6/2 mice, were still sensitive to cannabinoid receptor stimulation.

Animals↗
PubMed2005

Structural stability of soybean lipoxygenase-1 in solution as probed by small angle X-ray scattering.

Soybean lipoxygenase-1 (LOX-1) is used widely as a model for studying the structural and functional properties of the homologous family of lipoxygenases. The crystallographic structure revealed that LOX-1 is organized in a beta-sheet N-terminal domain and a larger, mostly helical, C-terminal domain. Here, we describe the overall structural characterization of native unliganded LOX-1 in solution, using small angle X-ray scattering (SAXS). We show that the scattering pattern of the unliganded enzyme in solution does not display any significant difference compared with that calculated from the crystal structure, and that models of the overall shape of the protein calculated ab initio from the SAXS pattern provide a close envelope to the crystal structure. These data, demonstrating that LOX-1 has a compact structure also in solution, rule out any major motional flexibility of the LOX-1 molecule in aqueous solutions. In addition we show that eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase used to mimic the effect of substrate binding, does not alter the overall conformation of LOX-1 nor its ability to bind to membranes. In contrast, the addition of glycerol (to 5%, v/v) causes an increase in the binding of the enzyme to membranes without altering its catalytic efficiency towards linoleic acid nor its SAXS pattern, suggesting that the global conformation of the enzyme is unaffected. Therefore, the compact structure determined in the crystal appears to be essentially preserved in these various solution conditions. During the preparation of this article, a paper by M. Hammel and co-workers showed instead a sharp difference between crystal and solution conformations of rabbit 15-LOX-1. The possible cause of this difference might be the presence of oligomers in the rabbit lipoxygenase preparations.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed2005

Radiochromatographic assay of N-acyl-phosphatidylethanolamine-specific phospholipase D activity.

A radiochromatographic method has been set up to assay the activity of N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), based on reversed-phase high-performance liquid chromatography (HPLC) and online scintillation counting. The anandamide (N-arachidonoylethanolamine, AEA), product released by NAPE-PLD from the N-arachidonoyl-phosphatidylethanolamine (NArPE) substrate, was separated using a C18 column eluted with methanol-water-acetic acid and was quantified with an external standard method. Baseline separation of AEA and NArPE was completed in less than 15 min, with a detection limit of 0.5 fmol AEA at a signal-to-noise ratio of 4:1. The sensitivity and accuracy of the radiochromatographic procedure allowed detection and characterization of NAPE-PLD activity in very tiny tissue samples or in samples where the enzymatic activity is very low. With this method, we could determine the kinetic constants (i.e., apparent Michaelis-Menten constant (Km) of 40.0+/-5.6 microM and maximum velocity (Vmax) of 22.2+/-3.5 pmol/min per milligram protein toward NArPE) and the distribution of NAPE-PLD activity in brain areas and peripheral tissues of mouse. In addition, we could collect unprecedented evidence that compounds widely used in studies of the endocannabinoid system (e.g., AEA and congeners, receptor a(nta)gonists and inhibitors of AEA degradation) can also affect NAPE-PLD activity.

Animals↗
PubMed2005

Lipid rafts control signaling of type-1 cannabinoid receptors in neuronal cells. Implications for anandamide-induced apoptosis.

Several G protein-coupled receptors function within lipid rafts plasma membrane microdomains, which may be important in limiting signal transduction. Here we show that treatment of rat C6 glioma cells with the raft disruptor methyl-beta-cyclodextrin (MCD) doubles the binding efficiency (i.e. the ratio between maximum binding and dissociation constant) of type-1 cannabinoid receptors (CB1R), which belong to the rhodopsin family of G protein-coupled receptors. In parallel, activation of CB1R by the endogenous agonist anandamide (AEA) leads to approximately 3-fold higher [35S]GTPgammaS binding in MCD-treated cells than in controls, and CB1R-dependent signaling via adenylate cyclase, and p42/p44 MAPK is almost doubled by MCD. Unlike CB1R, the other AEA-binding receptor TRPV1, the AEA synthetase NAPE-PLD, and the AEA hydrolase FAAH are not modulated by MCD, whereas the activity of the AEA membrane transporter (AMT) is reduced to approximately 50% of the controls. We also show that MCD reduces dose-dependently AEA-induced apoptosis in C6 cells but not in human CHP100 neuroblastoma cells, which mirror the endocannabinoid system of C6 cells but are devoid of CB1R. MCD reduces also cytochrome c release from mitochondria of C6 cells, and this effect is CB1R-dependent and partly mediated by activation of p42/p44 MAPK. Altogether, the present data suggest that lipid rafts control CB1R binding and signaling, and that CB1R activation underlies the protective effect of MCD against apoptosis.

Animals↗