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J. Biol. Chem., Vol. 278, Issue 35, 32726-32732, August 29, 2003
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From the
Department of Biomedical Sciences,
University of Teramo, Piazza A. Moro 45, 64100 Teramo, Italy, the IRCCS C.
Mondino, Mondino-Tor Vergata-Santa Lucia Center for Experimental Neurobiology,
Via Ardeatina 306, 00179 Rome, Italy, and the
¶Department of Experimental Medicine and
Biochemical Sciences, University of Rome Tor Vergata, Via Montpellier 1, 00133
Rome, Italy
Received for publication, February 28, 2003 , and in revised form, June 10, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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270% over the untreated controls. Stimulation of
FAAH occurred through up-regulation of gene expression at transcriptional and
translational level and was specific. Indeed, neither the activity of the
anandamide-synthesizing N-acyltransferase and phospholipase D, nor
the activity of the anandamide transporter, nor the binding to cannabinoid
receptors were affected by progesterone under the same experimental
conditions. The activation of FAAH by progesterone was paralleled by a
decrease (down to 60%) of the cellular levels of anandamide and involved
increased nuclear levels of the transcription factor Ikaros. Analysis of the
FAAH promoter showed an Ikaros binding site, and mutation of this site
prevented FAAH activation by progesterone in transient expression assays.
Electrophoretic mobility shift and supershift assays further corroborated the
promoter activity data. Furthermore, the effect of progesterone on FAAH
promoter was additive to that of physiological amounts of leptin, which binds
to a cAMP response element-like site in the promoter region. Taken together,
these results suggest that progesterone and leptin, by up-regulating the FAAH
promoter at different sites, enhance FAAH expression, thus tuning the
immunomodulatory effects of anandamide. These findings might also have
critical implications for human fertility. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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), thus allowing the survival of fetal
allograft and therefore a successful pregnancy
(10,
11). In this context, the
activity of FAAH in maternal peripheral lymphocytes is under control of
progesterone, which binds to its intracellular receptor and activates FAAH
expression (12). As a
consequence, the release of cytokines critical for fertility, such as the
leukemia inhibitory factor, is favored
(12). Moreover, lymphocyte
FAAH has been shown to control the levels of blood AEA in pregnant women,
where low FAAH activity implies high AEA levels, leading to spontaneous
abortion (7,
12,
13). Taken together, these
data have suggested a cross-talk between steroid hormones, cytokines, and the
peripheral endocannabinoid system in lymphocytes, which is implicated in
regulating immunity and fertility in humans
(14). Also leptin (L), the
16-kDa non-glycosylated product of the obese gene, serves systemic
functions, which include regulation of fertility
(15) and modulation of immune
response (16). Recently, L has
been shown to reduce the levels of AEA in the hypotalamus of ob/ob
mice, suggesting that this compound partakes of the neural circuitry regulated
by L (17). In addition, L
enhances FAAH expression by activating a CRE (cAMP response element)-like site
in the promoter region
(18).
Based on this background, we sought to investigate whether P might regulate AEA metabolism in human T lymphocytes. In fact, we show an enhancement of FAAH activity and expression by P, triggered through overexpression of the Ikaros (Ik) transcription factor (19, 20) and subsequent Ik-dependent up-regulation of promoter activity. We also show that the effect of P is synergistic with that of L.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Isolation and Treatment of T Lymphocytes—Blood samples (20 ml per donor) were drawn from the antecubital vein of healthy donors (age range 28–35 years), who gave informed consent to the study, and were collected into heparinized sterile tubes. Clearance of the local Ethics Committee was obtained to sue the human cells. Peripheral lymphocytes were purified by gradient centrifugation, using the density separation medium Lymphoprep (Nycomed Pharma, Oslo, Norway), and then T-cells were isolated from the whole lymphocyte population by means of the Dynal CD2 CELLection kit (Dynal, Olso, Norway), as reported previously (12). Purified T lymphocytes were resuspended in RPMI 1640 medium (Invitrogen, Paisley, UK), supplemented with 25 mM Hepes, 2.5 mM sodium pyruvate, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal bovine serum (Invitrogen), at a density of 1.5 x 106 cells/ml in ventilated 25-ml flasks (12). Incubation of T lymphocytes with P, alone or in the presence of different compounds, was performed at 37 °C in humidified 5% CO2 atmosphere, at the indicated concentrations and for the indicated periods of time. Cells were treated for 1 h in serum-free medium, and then heat-inactivated fetal bovine serum was added at a final concentration of 10% (12). Controls were incubated with vehicles alone. Cell viability after each treatment was tested by trypan blue dye exclusion and was found to be higher than 90% in all cases.
FAAH Activity and Expression—FAAH (arachidonoylethanolamide amidase; EC 3.5.1.4 [EC] ) activity was assayed at pH 9.0 with 10 µM [3H]AEA as substrate, by the reversed phase high performance liquid chromatography method described previously (18). FAAH activity was expressed as pmol of arachidonate released per min per mg of protein. Cell homogenates (20 µg/lane) were prepared as described previously (18) and were subjected to enzyme-linked immunosorbent assay (ELISA), to quantify FAAH protein. Wells were coated with human T-cell homogenates (20 µg/well), which were then reacted with anti-FAAH polyclonal antibodies (diluted 1:300), as first antibody, and with GAR-AP, diluted 1:2000, as second antibody (12). In a previous study, the anti-FAAH antibodies have been shown to recognize a single band in human lymphocytes by Western blot analysis (12). Color development of the alkaline phosphatase reaction was measured at 405 nm, using p-nitrophenyl phosphate as substrate. The A405 values could not be converted into FAAH concentrations, because the purified enzyme is not available to make calibration curves. However, the ELISA test was linear in the range 0–50 µg/well of cell homogenate, and its specificity for FAAH was validated by antigen competition experiments (12). Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using total RNA isolated from human T lymphocytes (10 x 106 cells) by means of the S.N.A.P.TM total RNA isolation kit (Invitrogen), as described previously (12). RT-PCR amplification of FAAH was performed using 100 ng of total RNA, and the EZ rTth RNA PCR kit (PerkinElmer Life Sciences), as reported previously (12). The amplification parameters were as follows: 2 min at 95 °C, 45 s at 95 °C, 30 s at 55 °C, and 30 s at 60 °C. Linear amplification was observed after 20 cycles. The primers for FAAH were as follows: (+), 5'-TGGAAGTCCTCCAAAAGCCCAG; (–), 5'-TGTCCATAGACACAGCCCTTCAG. Five µl of the reaction mixture were electrophoresed on a 6% polyacrylamide gel, and the RT-PCR products were excised from the gel and counted in a LKB1214 Rackbeta scintillation counter (Amersham Biosciences) (12). Products were validated by size determination and sequencing, as described previously (12).
Analysis of N-Acyltransferase, Phospholipase D, Anandamide Uptake, and Cannabinoid Receptors—N-Acyltransferase (NAT) assay was performed as described previously (22), using 1,2-di[1-14C]palmitoylphosphatidylcholine (1 x 106 dpm/test) as substrate and measuring the formation of N-[14C]palmitoylphosphatidylethanolamines by high performance thin layer chromatography on silica gel plates (Sigma). 1,2-Dipalmitoyl-N-palmitoyl-phosphatidylethanolamine was used as standard, and NAT activity was expressed as pmol of N-palmitoylphosphatidylethanolamine formed per min per mg of protein (22). The activity of phospholipase D (PLD; EC 3.1.4.4 [EC] ) was assayed in T-cell homogenates by measuring the release of [14C]ethanolamine from 1,2-dioleoyl-3-phosphatidyl-[2-14C]ethanolamine (10 µM), as described previously (23). PLD activity was expressed as pmol of ethanolamine released per min per mg of protein. The uptake of 200 nM [3H]AEA by intact T lymphocytes (2 x 106/test) through AMT was studied as described previously (18) and was expressed as pmol of AEA taken up per min per mg of protein. For CBR studies, membrane fractions prepared from T lymphocytes (10 x 106) as reported previously (18) were quickly frozen in liquid nitrogen and stored at –80 °C for no longer than 1 week. These membrane fractions were used in rapid filtration assays with the synthetic cannabinoid [3H]CP55.940 (used at 400 pM), as reported previously (18). Unspecific binding was determined in the presence of 100 nM "cold" agonist (12, 18). Receptor binding was expressed as fmol of ligand bound per mg of protein.
Analysis of the Endogenous Levels of AEA and 2-Arachidonoyglycerol (2-AG)—The endogenous levels of AEA and 2-AG in human T lymphocytes (50 x 106/test) were assayed at NIAAA/National Institutes of Health by liquid chromatography/mass spectrometry, using an Agilent 1100 series LC-MSD equipped with a thermostated autosampler and column compartment, as reported previously (24). The liquid chromatography/mass spectrometry analysis was also corroborated by gas chromatography-electron impact mass spectrometry, performed in Rome as described previously (25).
Western Blot Analysis of Nuclear Levels of Ikaros—For the determination of Ik isoforms in T lymphocytes, nuclear extracts were prepared from T-cell suspensions (19), and aliquots (50 µg protein) were loaded onto 10% SDS-polyacrylamide gels and were electroblotted onto 0.45-µm nitrocellulose filters (Bio-Rad), as described previously (19). For immunodetection, anti-Ik antiserum was diluted 1:1000, and the second antibody (GAR-AP) was diluted 1:2000 (12). Protein content was normalized before loading onto the gel (18), and equal loading of extracts was verified by Ponceau staining (19). Rainbow molecular mass markers (Amersham Biosciences) were bovine serum albumin (66.0 kDa) and ovalbumin (46.0 kDa). Nuclear levels of total Ik isoforms were further quantified by ELISA, performed by coating each well with 25 µg protein/sample, as described above for FAAH, and then reacted with anti-Ik antiserum (1:1000) and GAR-AP (1:2000).
Construction of Chloramphenicol Acetyltransferase Expression Vectors
and Transient Transfection—Sequence information for the upstream
regulatory region of FAAH gene was downloaded from GeneBankTM
(region: gi/11423254:644582-754250, International Human Genome Project), and
the proximal promoter region of base pairs from +1 to –107 (+1 being the
first nucleotide of the FAAH mRNA) was assembled using synthetic
oligonucleotides (Amersham Biosciences). The DNA was gel-purified and
subcloned into the PstI/XbaI sites of pCAT3-Basic vector
(Promega Corp., Madison, WI). The same strategy was used to introduce
mutations in the recombinant plasmids bearing the promoter region. The
nucleotide sequences of all constructs were verified by dideoxynucleotide
chain termination sequencing with a Sequenase kit 2.0 (United States
Biochemical, Cleveland, OH). Human T-cells (1 x 106 per test)
were transfected in triplicate using TransFastTM transfection reagent
(Promega Corp.), according to the manufacturer's instructions. Typically,
cells were washed in phosphate-buffered saline and resuspended in 0.5 ml of
serum-free medium, and then they were mixed with 0.5 ml of serum-free medium
containing 2 µg of total DNA and the TransFastTM transfection reagent,
at a charge ratio of 1:1 with respect to DNA. Transfection efficiency was
monitored by use of 0.5 µg of thymidine kinase 
-galactosidase
construct (Clontech, Palo Alto, CA). After transfection, the medium was
replaced with complete growth medium, and cells were harvested 48 h later. For
chloramphenicol acetyltransferase (CAT) activity assays, cellular extracts
were prepared as described above for FAAH, and different aliquots were used
for CAT assays, for -galactosidase activity determination, a marker of
transfection efficiency, and for protein quantitation. CAT activity was
determined using the Quan-T-CAT assay system (Amersham Biosciences), whereas
the activity of -galactosidase was assayed using the
-galactosidase enzyme system (Promega Corp.). The values of CAT activity
were normalized to -galactosidase activity and to the protein content
(18).
Nuclear Extracts, Electrophoretic Mobility Shift, and Supershift Assays—Nuclear extracts were prepared according to Schreiber et al. (26) with the modifications reported by Lee et al. (27). Electrophoretic mobility shift assay (EMSA) experiments were performed as described previously (27), using the Ikaros oligonucleotide –76 5'-AGGCGGGCGTGGGATCCCGGCTG-3'–54 (site in bold), whereas the oligonucleotides used for the cold competitions were 5'-CTCGCAGCCTGGGAAGATAAGTGG-3' (Ikaros site derived from vasoactive intestinal peptide receptor-1 promoter), and –76 5'-AGGCGGGCGTTTTTTCCCGGCTG-3'–54, which is the mutated site used for the transfection experiments (the mutated nucleotides are underlined) (28). In all oligonucleotides, the numbers refer to positions in the FAAH promoter. The complexes were resolved on non-denaturing 6% polyacrylamide gels in 0.5 x TBE buffer (0.45 M Tris borate, 10 mM EDTA, pH 8.0) for 1 h at 14 V/cm and were autoradiographed overnight. For gel supershift analysis, nuclear extracts were preincubated overnight at 4 °C with 3 µg of rabbit anti-Ik antiserum, before addition of 32P-labeled oligoucleotide (26). Dye was omitted from the loading buffer, and the gel was run at 4 °C in 0.2 x TBE buffer at 5 V/cm. The autoradiographic films were subjected to densitometric analysis by means of a Floor-S MultiImager equipped with Quantity One software (Bio-Rad), as reported previously (18).
Statistical Analysis—Data reported in this paper are the mean (±S.D.) of at least three independent determinations, each in duplicate. Statistical analysis was performed by the non-parametric Mann-Whitney test, elaborating experimental data by means of the InStat 3 program (GraphPAD Software for Science, San Diego, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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NAT, PLD, AMT, and CBR Are Not Affected by Treatment of T Cells with Progesterone—NAT catalyzes the biosynthesis of N-arachidonoylphosphatidylethanolamines by transferring an arachidonate group from the sn-1 carbon of phospholipids to the amino group of phosphatidylethanolamines (22). Together with the N-acylphosphatidylethanolamines (NAPE)-hydrolyzing PLD (23), NAT is considered the checkpoint in AEA synthesis (22–25). The activity of NAT and that of PLD were almost identical in T-cells untreated or treated with 1 µM P (Fig. 1C). Although the activity of PLD was assayed under conditions found to be optimal for the NAPE-hydrolyzing enzyme (23), a radiolabeled phosphatidylethanolamine was used as substrate instead of radiolabeled NAPEs, which are not yet commercially available (25). To date there is no evidence that this substrate is specific for the NAPE-hydrolyzing PLD, and there are no specific inhibitors of this enzyme that might allow to further extend its analysis and to conclusively assess its contribution to AEA metabolism. However, the lack of effect of P on the activity of both NAT and PLD in T lymphocytes seems to speak in favor of a lack of effect on AEA synthesis. Also the activity of AMT was not affected by 1 µM P (Fig. 1C), suggesting that the cellular uptake of AEA was not coupled to its breakdown by FAAH (12, 30). In this context, it seems noteworthy that the relationship between AMT and FAAH is still under debate, because FAAH might not quite need a transporter to get in contact with AEA (31), and AMT might work "in reverse" to export (rather than import) AEA (32). Human lymphocytes have CB receptors (12) and treatment with 1 µM P for 24 h did not affect their ability to bind [3H]CP55.940 through these receptors (Fig. 1C). Taken together with the data on FAAH, these results show for the first time that P down-regulates the tone of endocannabinoids in human T lymphocytes by activating their degradation by FAAH, rather than by reducing their synthesis through NAT and PLD or transport through AMT. To further elucidate the modulation of FAAH expression by P, we investigated the properties of the FAAH promoter in human T lymphocytes.
Analysis of the FAAH Promoter—The human FAAH gene
has been located on chromosome 1
(33), which has been
completely sequenced. Therefore, we inspected this chromosome to gain insight
into the FAAH promoter features. Human FAAH promoter was found to lack TATA
boxes, and like many genes bearing this feature, it had a proximally
positioned SP1 site (Fig.
2A). Moreover, there was another SP1 site in the reverse
orientation 100 nucleotides upstream
(Fig. 2A), a feature
that resembles the structure of the mouse proximal promoter
(34). P had no effect on the
FAAH minimal promoter (Fig. 3,
min), but it up-regulated the reporter CAT gene if under
control of the region between the two SP1 sites of the FAAH upstream region
(Fig. 3, wt). RU486
(10 µM) fully reverted the effect of 1 µM Pon CAT
activity (Fig. 3). Moreover,
under the same conditions that enhanced FAAH activity, P dose-dependently
increased the nuclear amount of Ikaros isoforms Ik1, Ik2, and Ik3, as revealed
by Western blot analysis (Fig.
4A). A further quantitative analysis of the nuclear
content of total Ik isoforms by ELISA showed that their level increased up to
220% of the controls in the nuclei of T lymphocytes treated for 24 h with
1 µM P (Fig.
4B). The effect of 1 µM P was fully
reverted by 10 µM RU486 (Fig.
4, A and B). On the other hand, L had no effect
on nuclear Ik content under the same experimental conditions
(Fig. 4B). Based on
these data, we searched for an Ikaros consensus site in the FAAH promoter and
found it at position –66 (Fig.
2A). Transient transfection experiments revealed that
this site is indeed functional, since its mutation abolished FAAH
up-regulation by P (Fig. 3,
mutIk). Moreover, EMSA experiments showed that T-cell nuclear
extracts formed a retarded complex in untreated cells which was more abundant
in cells treated for 24 h with 1 µM P, when an oligonucleotide
corresponding to the Ik site found at position –66 of the FAAH promoter
was used (Fig. 2B).
Specificity of the binding to the Ikaros site was confirmed by using a cold
competitor, which corresponds to an established functional Ik site derived
from the vasoactive intestinal peptide receptor-1 promoter
(28), and the mutated site
used for transient transfection experiments. Cold competitor and mutated site,
respectively, abolished or had no effect on the retarded complex
(Fig. 2B). The complex
was further retarded by using antibodies directed against the Ik isoforms in
gel supershift assays, thus confirming the identity of the protein binding to
the Ik site (Fig. 2B).
Densitometric analysis of gel shift and supershift experiments strengthened
the hypothesis that Ik was directly involved in the regulation of FAAH
expression (Fig. 2B).
In addition, transient expression experiments showed that P can activate FAAH
promoter in synergism with L, indeed a combination of 1 µM P and
10 nM L increased CAT activity up to 650% of the controls,
whereas they increased CAT up to 400% when used alone
(Fig. 3). In this context, it
should be recalled that 10 nM L has been recently shown to activate
the FAAH promoter in T-cells through a CRE-like site
(18), which was located
downstream of the Ik site in the promoter region
(Fig. 2A). Consistent
with these data, treatment of human lymphocytes for 24 h with 1
µM P + 10 nM L increased FAAH activity, protein
content, and mRNA level up to 570, 530, and 550% of the
untreated controls, respectively, demonstrating that L further potentiated the
effect of P on FAAH expression (Table
II).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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P stimulates FAAH activity and expression in T-cells at the same
circulating levels (from 0.02 to 0.30 µg/ml) found in pregnant women
(10). These data extend to T
lymphocytes our previous observation that P, in the same concentration range
used here, up-regulates FAAH gene expression in the whole population of
peripheral lymphocytes, by binding to its intracellular receptor
(12). In the same line, RU486,
a synthetic antiprogestinic which stabilizes progesterone receptor in a form
unable to bind DNA (29), fully
reverts the effect of P on FAAH (Fig. 1,
A and B). Remarkably, RU486 was effective at a
concentration (10 µM) known to modulate immuno-endocrine
interactions in early pregnancy of humans
(35). On the other hand, P was
ineffective on NAT, PLD, and AMT activity, and on CBR binding in T lymphocytes
(Fig. 1C), suggesting
that FAAH was the only checkpoint for the effect of this hormone. These
observations are consistent with a role for FAAH in modulating immunoendocrine
interactions in early pregnancy in humans
(7,
12). They are also in keeping
with the hypothesis that FAAH is the key-regulator of AEA levels in
vivo; indeed, FAAH knock-out mice show 15-fold higher levels of AEA
than wild-type littermates
(36), and AEA levels in human
blood inversely correlate with FAAH activity in peripheral lymphocytes
(13). In this line, we show
that 1 µM P, which increased FAAH in T lymphocytes up to
270% over controls (Fig.
1A), reduced AEA levels in these cells down to 60%
(Table I). Interestingly, FAAH
has been shown to be critically linked to drug/alcohol abuse and dependence in
humans (37), again suggesting
that this enzyme is pivotal for controlling the biological activity of AEA and
potentially that of other FAAH-hydrolyzable congeners like 2-AG
(17). At any rate, the
up-regulation of FAAH expression by P in human T-cells is a major finding of
this investigation, associated with higher FAAH activity and lower AEA content
in these cells.
The mechanism of FAAH activation by P was further investigated by analyzing
the FAAH promoter. Like many promoters lacking a TATA box, it had a proximally
positioned SP1 site, which was preceded, both in the human and mouse
promoters, by another SP1 site in reverse orientation
(Fig 2A). FAAH
promoter also revealed a binding site for Ikaros, a member of the Kruppel
family of "zinc finger" DNA-binding proteins, which acts as a
critical transcriptional regulator of lymphocyte ontogeny and differentiation
(20). Ikaros comprises eight
different isoforms, Ik1 through Ik3 localizing to the nucleus and Ik4 through
Ik8 localizing to the cytosol
(20). Recently Ik1, Ik2, and
Ik3, the only isoforms that exhibit high DNA binding affinity
(20), have been detected in
the nucleus of human peripheral blood mononuclear cells
(19,
20), where they are essential
in controlling the activation of granzyme B promoter by dexamethasone
(19). Interestingly, we found
that human T-cells express nuclear Ik1, Ik2, and Ik3
(Fig. 4A) and that the
same doses of P that enhanced FAAH also increased up to 220% over the
controls the nuclear levels of these Ikaros isoforms
(Fig. 4B). Homo- and
heterodimer formation between Ik1, Ik2, and Ik3 is known to greatly increase
their affinity for DNA and their consequent ability to activate transcription
(19,
20). Therefore, it can be
concluded that P activates FAAH in human T lymphocytes, by stimulating the
binding of Ikaros to DNA, thus enhancing its transcriptional activity on the
FAAH promoter. Transfection experiments using FAAH promoter constructs with
mutated Ikaros (mutIk) revealed that indeed this site confers P responsiveness
(Fig. 3), and EMSA and gel
supershift analysis further corroborated this conclusion
(Fig. 2B). In
addition, we show unprecedented evidence that the effect of P on FAAH promoter
may be synergistic with that of physiological concentrations of L
(Fig. 3), which up-regulates a
CRE-like element downstream of the Ikaros site in the promoter region
(Fig. 2A).
It seems noteworthy that to date only one report has described the modulation of human FAAH promoter by L (18). Two other interesting reports have characterized the promoter (38) and the transcriptional regulation (34) of mouse FAAH in neuronal cell lines. In particular, they have shown either putative (38) or imperfect (34) estrogen response elements in the FAAH promoter region, giving some ground to our previous report that estrogen down-regulates FAAH activity in mouse (39). Here, we show that in the human FAAH promoter estrogen response elements are replaced by an Ikaros binding site (Fig. 2A). In this context, we have recently shown that estrogen down-regulates FAAH in human cells according to a nongenomic mechanism (32). These observations suggest a relevant species specificity of FAAH regulation, although the human and mouse FAAH (localized on chromosomes 1 and 4, respectively) share 84% sequence identity (21) and have a conserved genomic structure (33). In addition, also a tissue specificity of FAAH promoter activity has been observed (38), which might further contribute to divergent regulation in different species or in different tissues of the same species. The interaction between different transcription factors, some of which have been identified here for the first time, on FAAH regulation awaits for further clarification.
In conclusion, the results reported here represent the first evidence of a link between the hormone-cytokine networks controlled in T lymphocytes by progesterone (11) and/or leptin (40), and the peripheral endocannabinoid system, and suggest that AEA hydrolysis by FAAH might be the target for new therapies of human defects in immunity and fertility.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Biomedical Sciences,
University of Teramo, Piazza A. Moro 45, 64100 Teramo, Italy. Tel.:
39-0861-266875; Fax: 39-0861-412583; E-mail:
Maccarrone{at}vet.unite.it.
1 The abbreviations used are: AEA, anandamide
(N-arachidonoylethanolamine); 2-AG, 2-arachidonoylglycerol; AMT, AEA
membrane transporter; CAT, chloramphenicol acetyltransferase; CBR, cannabinoid
receptors; CP55.940,
5-(1,1'-dimethyheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl)cyclohexyl]phenol;
CRE, cAMP-response element; ELISA, enzyme-linked immunosorbent assay; EMSA,
electrophoretic mobility shift assay; FAAH, fatty acid amide hydrolase;
GAR-AP, goat anti-rabbit antibodies conjugated with alkaline phosphatase; Ik,
Ikaros; L, leptin; NAT, N-acyltransferase; P, progesterone; RT,
reverse transcriptase; PLD, phospholipase D; NAPE,
N-acylphosphatidylethanolamines.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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