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Prepublished online as a Blood First Edition Paper on August 1, 2002; DOI 10.1182/blood-2002-05-1444.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Experimental Medicine and
Biochemical Sciences, University of Rome Tor Vergata,
Italy.
Estrogen replacement therapy has been associated with reduction of
cardiovascular events in postmenopausal women, though the mechanism for
this benefit remains unclear. Here we show that at physiological
concentrations estrogen activates the anandamide membrane transporter
of human endothelial cells and leads to rapid elevation of calcium
(apparent within 5 minutes) and release of nitric oxide (within 15 minutes). These effects are mediated by estrogen binding to a surface
receptor, which shows an apparent dissociation constant
(Kd) of 9.4 ± 1.4 nM, a maximum binding (Bmax) of 356 ± 12 fmol × mg
protein Cardiovascular diseases are the leading cause of
death in postmenopausal women in developed countries, suggesting that
estrogen (17 Materials
Endothelial cell culture and coculture with human
platelets
Binding studies and Western blot analysis The isolation of nuclear pellets and nuclei-free cell membrane pellets from HUVECs was performed as reported.23 These 2 fractions were used in rapid filtration assays with [3H]E2 as described,24 and the binding data were elaborated through nonlinear regression analysis, using the Prism 3 program (GraphPAD Software for Science, San Diego, CA). In all binding experiments, nonspecific binding was determined in the presence of 1 µM "cold" E2.24 For Western blot analysis, nuclear or cell membrane homogenates (10 or 20 µg/lane, respectively) were prepared as described24 and were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12%), followed by electrotransfer to 0.45-µm nitrocellulose filters (Bio-Rad), as reported.25 Rainbow molecular weight markers (Amersham Pharmacia Biotech) were bovine serum albumin (66 kDa) and ovalbumin (46 kDa). Filters were immunoreacted with anti-ER or
anti-ER polyclonal antibodies and were diluted 1:200 using goat
antirabbit or donkey antigoat alkaline phosphatase conjugate (diluted
1:2000) as second antibody, respectively.24 The
specificity of the immunoreactive bands was assessed by preincubating anti-ER antibody (10 µg/mL) with the ER competing peptide (70 µM), as reported.25 Densitometric analysis of filters
was performed by means of a Floor-S Multi-Imager, equipped with
Quantity One software (Bio-Rad Laboratories).
Determination of anandamide uptake and release The uptake of [3H]AEA by intact HUVECs through the anandamide membrane transporter (AMT) was studied as described.7 Cell suspensions (1 × 106 cells/test) were incubated for different time intervals, at 37°C, with 100 nM [3H]AEA, and AMT activity was expressed as picomole AEA taken up per minute per milligram protein. Incubations (15 minutes) were also carried out with different concentrations of [3H]AEA, in the range of 0 to 1000 nM to determine apparent Km and Vmax of the uptake by Lineweaver-Burk analysis.7 The effect of different compounds on AEA uptake (15 minutes) was determined by adding each substance directly to the incubation medium at the indicated concentrations. The ability of HUVECs (5 × 106/test), untreated or pretreated with E2 and related compounds for 4 hours as described above, to release AEA into the culture medium was determined by loading cells with [3H]AEA (1 µCi/106 [0.037 MBq/106] cells) for 10 minutes14 in the presence of 10 µM ATFMK, then washing and measuring at different incubation times the radioactivity of the culture medium containing 10 µM ATFMK.15 The identity of AEA was ascertained by reverse-phase-high-performance liquid chromatography (RP-HPLC), as reported,26 yielding chromatograms like those shown in Figure 3C. In some experiments, HUVECs (1 × 106 cells/test) were preloaded with 100 µM cold AEA for 30 minutes and were washed, and then their ability to take up 100 nM [3H]AEA from the culture medium was determined as described above. A large-scale experiment was performed by incubating HUVECs (10 × 106/test) for 1 hour at 37°C with [3H]arachidonic acid (10 µCi/test [0.37 MBq/test]) in serum-free culture medium, containing or not containing 100 nM E2, and then washing and resuspending the cells in complete culture medium with 10 µM ATFMK for an additional 3 hours at 37°C. Membrane lipids were extracted as described previously,26 and radioactivity was measured in lipid extracts and culture media by liquid scintillation counting. Cell viability after each treatment was checked with trypan blue and was found to be higher than 90%.Enzymatic assays FAAH (E.C. 3.5.1.4) activity was assayed in HUVEC extracts by measuring the release of [3H]arachidonic acid from [3H]AEA, using RP-HPLC.26 Nitric oxide synthase (E.C. 1.14.13.39; NOS) was assayed by measuring the conversion of [3H]arginine into [3H]citrulline.27 15-lipoxygenase (E.C. 1.13.11.12; 15-LOX) was assayed by measuring the oxidation of [3H]linoleic acid to 13-hydroxyoctadecadienoic acid (13-HODE).28 Phospholipase D (E.C. 3.1.4.4; PLD) was assayed by measuring the release of [14C]ethanolamine from 1,2-dioleoyl-3-phosphatidyl-[2-14C]ethanolamine.29 The activities of cellular FAAH, NOS, 15-LOX, and PLD were within the linearity range of calibration curves drawn with partially purified FAAH (0-200 U/mg protein), purified NOS (0-250 mU/mg protein), purified soybean lipoxygenase-1 (0-10 mU/mg protein), 15-LOX,30 and purified PLD (0-1 mU/mg protein), respectively.Nitrite release and calcium levels in endothelial cells Generation of NO by endothelial cells (5 × 106 cells/test) was determined by measuring accumulation after 15 minutes of the stable end product nitrite (NO Determination of cAMP and IP3 levels in human platelets and binding of [3H]ADP Cyclic adenosine monophosphate (cAMP) levels in extracts of platelets (5 × 109/test) were determined by the Cayman Chemical cAMP Enzyme Immunoassay kit (Alexis), as reported.14 Inositol-1,4,5-trisphosphate (IP3) content in extracts of platelets (5 × 109/test) was measured by the IP3 [3H]Radioreceptor Assay kit (NEN Life Science Products, Boston, MA), as reported.14 The effect of AEA on the binding of 500 pM [3H]ADP to membrane fractions prepared from human platelets (10 × 1010/test), as described,14 was determined by rapid filtration assays.24 Unspecific binding was determined in the presence of 1 µM ADP.24Statistical analysis Data reported in this paper are the means ± SD of at least 3 independent experiments, each performed in duplicate. The initial velocities and the half-times of the in and out flux of AEA through AMT were calculated by fitting the time-course data to a sum of 2 exponential relaxation processes through MATLAB 5.2 software (The Mathworks). Statistical analysis was performed using the nonparametric Mann-Whitney U test, elaborating experimental data by means of the InStat 3 program (GraphPAD Software for Science).
Stimulation of AMT, nitric oxide, and calcium in endothelial cells by E2 HUVECs accumulate [3H]AEA through selective AMT showing an apparent Km of 190 ± 10 nM and an apparent Vmax of 45 ± 3 pmol × minute 1 × mg protein1.7 In
vitro treatment of HUVECs with E2 enhanced AMT activity in a
dose-dependent manner, as did E2 conjugated to BSA (Figure 1A). AMT activation by E2 or E2-BSA
reached statistical significance (P < .05) at 10 nM and
an approximately 4-fold maximum at 100 nM. Under the latter conditions,
kinetic analysis of [3H]AEA uptake by AMT showed an
apparent Km of 200 ± 10 nM and
Vmax of 225 ± 15 pmol × minute1 × mg protein1. Instead,
17-estradiol (epi-E2), the inactive epimer of E2,31 or
cortisol, which acts at glucocorticoid receptors,21 were ineffective at up to 200 nM concentration. The activation of AMT by 100 nM E2 or E2-BSA was further investigated in time-course experiments showing that either compound was effective
(P < .05) 30 minutes after HUVEC treatment and reached a
maximum at 4 hours (not shown). Treatment of HUVECs with E2 or E2-BSA
also enhanced NO release (Figure 1B) and intracellular calcium (Figure
1C) dose dependently; the calcium increase was apparent earlier (5 minutes) than the NO increase (15 minutes). NO and calcium increases
reached statistical significance (P < .05) at an E2 or
E2-BSA concentration of 10 nM and a 4- to 5-fold maximum at 100 nM.
Instead, treatment of HUVECs with epi-E2 or cortisol up to 200 nM was
ineffective (Figure 1B-C).
Binding of E2 to endothelial cell membranes HUVEC cell membranes were found to bind [3H]E2 with saturation curves like those shown in Figure 2A. From these curves, an apparent Kd of 9.4 ± 1.4 nM and a Bmax of 356 ± 12 fmol × mg protein1 could be calculated.
TMX (100 nM), an estrogen receptor antagonist,5,6 fully
displaced E2 from its membrane receptor, whereas 100 nM ICI182780, a
selective antagonist of nuclear estrogen receptors,5,6 was
ineffective (Figure 2A). E2 was able to bind to a nuclear receptor in
HUVECs through a saturable process (not shown) with Kd and Bmax values of 3.4 ± 0.3
nM and 2067 ± 37 fmol × mg protein1. TMX and
ICI182780 (each used at 100 nM) fully displaced this binding.
Furthermore, cold E2 (1 µM) fully displaced [3H]E2 from
membrane and nuclear receptors. Interestingly, the
Kd value of the HUVEC nuclear receptor for E2 is
close to that of authentic estrogen receptor (ER), whereas ER
has a Kd approximately 10-fold
lower.23 Consistent with the binding data, Western blot analysis showed that anti-ER, but not anti-ER, antibodies
recognized a single immunoreactive band in HUVEC extracts (Figure 2A
inset and data not shown), with the expected molecular mass for ER (approximately 60 kDa).23,24 This band disappeared when
anti-ER antibodies were preincubated with the competing peptide (not
shown). Densitometric analysis of filters like those shown in Figure 2A (inset) suggested that the level of membrane receptors was
approximately 6-fold lower than that of nuclear receptors. Taken
together with the binding data, it can be suggested that HUVECs possess
a nuclear ER that has a higher affinity for E2 and is expressed at
higher level than the estrogen surface receptor (ESR). This hypothesis is corroborated by the transcription of the gene of ER, but not of
ER, reported in HUVECs.5
Modulation of E2-induced activation of AMT by various compounds The activation of AMT by E2 was further investigated by using the agonists, antagonists, and inhibitors listed in Table 1. The effect of 100 nM E2 or E2-BSA was fully inhibited by 100 nM TMX, whereas at the same concentration ICI182780 had no effect (Figure 2B). HUVECs express a functional CB1 receptor,7,37 and 100 nM HU-210 increased AMT activation to approximately 160% of the estrogen-treated cells that is,
approximately 700% of the untreated controls (Figure 2B). Remarkably,
100 nM HU-210 alone has been shown to increase AMT activity in HUVECs
only to approximately 140% of the untreated controls.7
Conversely, SR141716, CBD, and CAPS did not affect AMT activation by
100 nM E2 or E2-BSA when used at 2 µM (Figure 2B). Furthermore,
abnormal-CBD and capsaicin were ineffective up to 2 µM (not shown).
AMT activation by 100 nM E2 or E2-BSA was also abolished by 400 µM
L-NAME or by 50 µM EGTA-AM, a cell-permeant
calcium chelator (Figure 2B).24 On the other hand, 100 nM
TMX fully inhibited the enhancement of NO release and calcium induced
by 100 nM E2, whereas 100 nM HU-210 further increased NO release to
160% of the E2-treated cells (Figure 2C). ICI182780 (100 nM),
SR141716, CBD, CAPS, abnormal-CBD, and capsaicin (each used at 2 µM)
were ineffective on NO and calcium levels (Figure 2C and data not
shown). Moreover, 400 µM L-NAME prevented E2-induced NO
release without affecting calcium levels, whereas 50 µM
EGTA-AM fully prevented calcium elevation and reduced NO
release to 45% of the E2-treated cells (Figure 2C).
AMT of endothelial cells is a bidirectional transporter HUVECs preloaded with [3H]AEA were able to release it in the culture medium in a time-dependent manner (Figure 3A). Release of [3H]AEA at 20 minutes was fully prevented by 10 µM AM404, and kinetic analysis of the release process showed a t1/2 of 5 ± 1 minute and an initial rate (v0) of 20 ± 2 pmol × minute1 × mg protein1.
The same kinetic analysis of the time-course of the
[3H]AEA uptake yielded
t1/2 = 5 ± 1 minute and
v0 = 23 ± 2 pmol × minute1mg
protein1, confirming our previous data.7 E2
and E2-BSA dose dependently enhanced [3H]AEA release from
HUVECs, reaching statistical significance (P < .05) at 5 nM and an approximately 5-fold maximum at 100 nM, whereas epi-E2 or
cortisol were ineffective at concentrations up to 200 nM (Figure 3B).
RP-HPLC analysis demonstrated that the released compound was indeed AEA
(Figure 3C). From calibration curves (Figure 3C, inset), it could be
estimated that 100 nM E2 led to an AEA release of 1500 ± 150
fmol/106 cells, compared with 300 ± 30
fmol/106 cells of controls. Table
2 shows that 100 nM TMX prevented
E2-stimulated release of AEA from HUVECs, whereas 100 nM HU-210
increased it to 160% of the E2-treated cells. Again SR141716, CBD,
CAPS, abnormal-CBD, and capsaicin (each used at 2 µM) were
ineffective (Table 2 and data not shown). Instead, stimulation of AEA
release by 100 nM E2 was fully prevented by 10 µM AM404, 400 µM
L-NAME, or 50 µM EGTA-AM (Table 2).
The ability of AMT to transport AEA across membranes
bidirectionally was further investigated by preloading HUVECs with cold AEA and measuring the uptake of [3H]AEA from the
extracellular medium. Obviously, a reversible carrier must be able to
bind the solute on both sides of the membrane, and a high concentration
of the solute on one side should favor the exchange of intracellular
with extracellular solute molecules, leading to the so-called
trans effect of flux coupling.19 Such a
trans effect experiment demonstrated that indeed HUVECs were able to accumulate [3H]AEA at a rate of 15 ± 2
pmol × minute
E2 modulates the enzymes responsible for AEA metabolism in endothelial cells Treatment of HUVECs with 100 nM E2 or E2-BSA decreased FAAH activity (down to 20% of the controls) and increased the activity of NOS, 15-LOX, and PLD (by 400%, 320%, and 275% of the controls, respectively) (Table 3). PLD was assayed under the optimal conditions for N-acyl-phosphatidylethanolamines (NAPE)-hydrolyzing PLD,29 but a radiolabeled phosphatidylethanolamine was used instead of radiolabeled NAPEs, which are not commercially available. This seems noteworthy, because NAPE-hydrolyzing PLD is considered the checkpoint in AEA synthesis, though the lack of specific inhibitors of its activity makes it difficult to assess conclusively its contribution to AEA metabolism.10 The effect of E2 on FAAH, NOS, 15-LOX, or PLD activities was fully prevented by 100 nM TMX, whereas 100 nM ICI182780, 2 µM SR141716, 2 µM CBD, or 2 µM CAPS was ineffective (Table 3). On the other hand, NOS activity was further enhanced by 100 nM HU-210 up to 160% of the E2-treated cells (Table 3). L-NAME, ATFMK, and ETYA almost completely inhibited the activity of the target enzymes in HUVECs, whereas suramin and ST638 reduced cellular PLD to approximately 55% of controls (Table 3). The inhibition of 15-LOX by 10 µM ETYA relieved almost completely the inhibition of FAAH by 100 nM E2 (Table 3), suggesting that 15-LOX activity was involved in this inhibition. EGTA-AM reduced the activity of NOS, 15-LOX, and PLD to 42%, 47%, and 49% of the same activities in E2-treated cells, but not that of FAAH (Table 3), probably because NOS,27 15-LOX,30 and PLD39 depend on calcium for their activity, as does NAPE-hydrolyzing PLD in blood cells.40Coculture with E2-treated HUVECs inhibits serotonin release from human platelets In pilot experiments it was found that human platelets do not release detectable amounts of [3H]5-HT when cultured for 10 minutes in serum-free EGM-2 Bulletkit medium. However, they release 300 ± 30 fmol/109 cells on stimulation for 2 minutes with 1 µM ADP, a physiological platelet agonist, in keeping with previous reports.41,42 Endothelial cells, pretreated for 4 hours with 100 nM E2, reduced to 50% the release of [3H]5-HT from ADP-stimulated human platelets, whereas control HUVECs had no effect (Figure 4A). Instead, HUVECs pretreated with E2 but in the presence of 100 nM TMX or of 100 U/mL FAAH had no effect on the release of [3H]5-HT from ADP-stimulated platelets, nor did 100 nM E2 or E2-BSA added directly to the medium during the 10-minute coculture period (Figure 4A). A dose-dependent decrease of [3H]5-HT release, reaching statistical significance (P < .05) at 50 nM AEA and a minimum (50%) at 100 nM, was observed on treatment of ADP-stimulated platelets for 10 minutes with AEA (Figure 4B). This effect of AEA was not affected by SR141716, SR144528, CBD, or CAPS, each used at 2 µM (Figure 4B). On the other hand, the AEA hydrolysis products arachidonic acid and ethanolamine were ineffective at 100 nM. Platelet membranes were able to bind 500 pM [3H]ADP in rapid filtration assays, and AEA displaced 35% of bound [3H]ADP when used at 100 nM (P < .05 compared with controls), whereas the 5-HT transporter of human platelets43 was not affected by AEA at concentrations up to 10 µM (unpublished results).
Treatment of platelets with 1 µM ADP decreased intracellular cAMP
levels from 2.4 ± 0.2 to 1.2 ± 0.1 pmol × mg
protein
In this study we have shown that E2 activates endothelial AMT
through calcium and NO-dependent mechanisms. These effects are mediated
by the binding of E2 to a surface receptor that has a different
affinity for E2 but the same molecular mass as the nuclear ER The activation of AMT was observed at physiological concentrations of
E2,5,6,31 was rapid (30 minutes), and was mediated by a
specific E2 surface receptor (ESR), as demonstrated by its inhibition
by TMX, by its insensitivity to ICI182780, and by the lack of effect of
epi-E2 or cortisol. Consistent with this hypothesis, E2-BSA, which is
too large to penetrate the cell membrane, also activated AMT. The
structural nature of ESR remains a complicated issue; however, we
speculate that it might be similar to nuclear ER A major finding of this investigation is that AMT can transport AEA across the membrane in both directions, as shown by uptake and release experiments (Figure 3; Table 2) and as supported by the trans effect test. Kinetic analysis of AEA uptake and release and the effect of the AMT inhibitor AM404 strongly suggest that the in-and-out movement of AEA occurred through the same transporter. E2 enhanced AEA release in the same TMX-, HU-210-, L-NAME-, and EGTA-AM-sensitive and the same ICI182780-, SR141716-, CBD-, and CAPS-insensitive manner (Table 2) observed with AEA uptake (Figure 2B). Because E2, again in a TMX-sensitive and an ICI182780-insensitive manner, reduced the activity of the AEA-hydrolyzing enzyme FAAH and increased the activity of the AMT-stimulating enzyme NOS and of the AEA-synthesizing enzyme PLD (Table 3), it is reasonable to conclude that the biologic action of E2 is to enhance the release rather than the uptake of this lipid. This unprecedented observation of a physiological stimulus for AMT to act in reverse explains the reports of a retrograde signaling mediated by endocannabinoids in the brain, based on a calcium-dependent release of AEA from postsynaptic neurons (for a review, see MacDonald and Vaughan46 and the references cited therein). A further point of interest is that E2 stimulates 15-LOX in HUVECs, whereas the inhibition of this enzyme by ETYA reverses the effects of E2 on FAAH (Table 3). Thus it appears that lipoxygenase activity is responsible for the inhibition of FAAH induced by E2. An E2-mediated release of AEA from HUVECs might lead to activation of CB1 receptors by AEA itself, followed by NOS activation (Table 3) and further AEA release (Table 2), by an autocrine mechanism. The effect of the synthetic CB1 agonist HU-210 strongly supports this hypothesis. Once released within the blood vessels, AEA can exert its manifold
actions on the cardiovascular system, spanning from vasodilation and
related hypotension and bradycardia (for a review, see Kunos et
al12) to modulation of cell migration16 and
of immune response.18 Interestingly, E2 also regulates the
immune system.47 Another effect of E2 is on
platelets,1 which play a critical role in the pathogenesis
of atherosclerosis and cardiovascular disease3 and express
an intracellular E2 receptor.4 Platelets share several
features with neurons and respond to
endocannabinoids,13,14 thus representing a useful model
for the less accessible central nervous system. It is known that
endocannabinoids can modulate serotonergic transmission.48
For instance, AEA has recently been shown to decrease 5-HT levels in
the hippocampus.49 Here it is shown for the first time
that the secretion of 5-HT from ADP-stimulated platelets is reduced by
a diffusible factor released by E2-stimulated HUVECs in a TMX-sensitive
manner, but not by E2 itself (Figure 4A). The effect of this factor is
abolished by FAAH (Figure 4A) and is reproduced by AEA added at
nanomolar concentrations (Figure 4B), similar to those found in
blood.50 These findings suggest that AEA released from
HUVECs on stimulation with E2 inhibits 5-HT secretion from stimulated
platelets. This effect of AEA does not involve classical (CB1 or CB2)
or nonclassical (endothelial-type) cannabinoid receptors or vanilloid
receptors in platelets; rather, it occurs through a partial antagonism
by AEA at ADP receptors (Figure 4B and data not shown). Because AEA does not affect IP3 levels or those of cAMP in platelets,
it can be speculated that AEA might interfere with the binding of ADP to the P2X receptor, which is independent of IP3 or cAMP,
at variance with the P2TAC or P2TPLC
receptors.44 However, because ADP binding to platelets is
complex, the interactions of endocannabinoids with ADP signaling
deserve further investigation. In our study it appeared that whatever
the signal transduction pathway(s), AEA reduced 5-HT secretion from
platelets, whereas E2 did not. On the other hand, E2 decreased
IP3 and increased cAMP levels in ADP-treated platelets,
which is consistent with reduced platelet activation,44
whereas micromolar concentrations of AEA did not (Figure 4C and data
not shown). Because E2 acts on platelets in a TMX- and
ICI182780-sensitive manner, whereas E2-BSA is ineffective (Figure 4C),
it can be suggested that it binds at an intracellular receptor.1,4 Moreover, the results suggest that AEA
released from endothelial cells on E2 stimulation might complement the biologic activity of E2 itself because it can directly inhibit platelet
aggregation and indirectly inhibit the release of 5-HT. The
cross-talk between endothelial cells and platelets can be even more
complex
In conclusion, the results reported here demonstrate that E2 activates the synthesis and inhibits the degradation of AEA in human endothelial cells by acting at a surface receptor and causing a calcium-dependent release of NO. As a consequence, AEA is released in blood, where it can modulate the cardiovascular and immune systems. In particular, AEA released from estrogen-stimulated endothelial cells is capable of reducing the secretion of 5-HT from platelets. This newly found interplay between estrogen and AEA metabolism suggests that endocannabinoids might mediate some of the beneficial effects of estrogen, and it seems to indicate a novel approach for the management of cardiovascular disease in postmenopausal women.
We thank Prof Francesco Malatesta (University of L'Aquila, Italy) for fitting the time-course data of the in-and-out movement of anandamide and Dr Marianna Di Rienzo for skillful assistance.
Submitted May 16, 2002; accepted July 12, 2002.
Prepublished online as Blood First Edition Paper, August 1, 2002; DOI 10.1182/blood-2002-05-1444.
Supported by Istituto Superiore di Sanità (III AIDS Program) and Consiglio Nazionale delle Ricerche (Biotechnology Program L. 95/95), Rome, Italy.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Mauro Maccarrone, Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Via Montpellier 1, I-00133 Rome, Italy; email: maccarrone{at}med.uniroma2.it.
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© 2002 by The American Society of Hematology.
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  M. L. Yates and E. L. Barker Inactivation and Biotransformation of the Endogenous Cannabinoids Anandamide and 2-Arachidonoylglycerol Mol. Pharmacol., July 1, 2009; 76(1): 11 - 17. [Abstract] [Full Text] [PDF]
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 M. G. Gervasi, M. Rapanelli, M. L. Ribeiro, M. Farina, S. Billi, A. M. Franchi, and S. P. Martinez The endocannabinoid system in bull sperm and bovine oviductal epithelium: role of anandamide in sperm-oviduct interaction Reproduction, March 1, 2009; 137(3): 403 - 414. [Abstract] [Full Text] [PDF]
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 A A Izzo and M Camilleri Emerging role of cannabinoids in gastrointestinal and liver diseases: basic and clinical aspects Gut, August 1, 2008; 57(8): 1140 - 1155. [Abstract] [Full Text] [PDF]
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 L. Adermark and D. M. Lovinger Retrograde endocannabinoid signaling at striatal synapses requires a regulated postsynaptic release step PNAS, December 18, 2007; 104(51): 20564 - 20569. [Abstract] [Full Text] [PDF]
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 A.H. Taylor, C. Ang, S.C. Bell, and J.C. Konje The role of the endocannabinoid system in gametogenesis, implantation and early pregnancy Hum. Reprod. Update, September 1, 2007; 13(5): 501 - 513. [Abstract] [Full Text] [PDF]
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 G. Rossi, V. Gasperi, R. Paro, D. Barsacchi, S. Cecconi, and M. Maccarrone Follicle-Stimulating Hormone Activates Fatty Acid Amide Hydrolase by Protein Kinase A and Aromatase-Dependent Pathways in Mouse Primary Sertoli Cells Endocrinology, March 1, 2007; 148(3): 1431 - 1439. [Abstract] [Full Text] [PDF]
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 H. Wang, S. K. Dey, and M. Maccarrone Jekyll and Hyde: Two Faces of Cannabinoid Signaling in Male and Female Fertility Endocr. Rev., August 1, 2006; 27(5): 427 - 448. [Abstract] [Full Text] [PDF]
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 M. Maccarrone, B. Barboni, A. Paradisi, N. Bernabo, V. Gasperi, M. G. Pistilli, F. Fezza, P. Lucidi, and M. Mattioli Characterization of the endocannabinoid system in boar spermatozoa and implications for sperm capacitation and acrosome reaction J. Cell Sci., October 1, 2005; 118(19): 4393 - 4404. [Abstract] [Full Text] [PDF]
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 O. M. H. Habayeb, A. H. Taylor, M. D. Evans, M. S. Cooke, D. J. Taylor, S. C. Bell, and J. C. Konje Plasma Levels of the Endocannabinoid Anandamide in Women--A Potential Role in Pregnancy Maintenance and Labor? J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5482 - 5487. [Abstract] [Full Text] [PDF]
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 M. Maccarrone, M. Di Rienzo, N. Battista, V. Gasperi, P. Guerrieri, A. Rossi, and A. Finazzi-Agro The Endocannabinoid System in Human Keratinocytes: EVIDENCE THAT ANANDAMIDE INHIBITS EPIDERMAL DIFFERENTIATION THROUGH CB1 RECEPTOR-DEPENDENT INHIBITION OF PROTEIN KINASE C, ACTIVATING PROTEIN-1, AND TRANSGLUTAMINASE J. Biol. Chem., September 5, 2003; 278(36): 33896 - 33903. [Abstract] [Full Text] [PDF]
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M. Maccarrone, M. Bari, M. Di Rienzo, A. Finazzi-Agro, and A. Rossi Progesterone Activates Fatty Acid Amide Hydrolase (FAAH) Promoter in Human T Lymphocytes through the Transcription Factor Ikaros: EVIDENCE FOR A SYNERGISTIC EFFECT OF LEPTIN J. Biol. Chem., August 29, 2003; 278(35): 32726 - 32732. [Abstract] [Full Text] [PDF]
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M. Maccarrone, M. Di Rienzo, A. Finazzi-Agro, and A. Rossi Leptin Activates the Anandamide Hydrolase Promoter in Human T Lymphocytes through STAT3 J. Biol. Chem., April 4, 2003; 278(15): 13318 - 13324. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
9-tetrahydrocannabinol by binding to central (CB1) and
peripheral (CB2) cannabinoid receptors.
-nitro-L-arginine methyl ester
(L-NAME), 5,8,11,14-eicosatetraynoic acid (ETYA), adenosine
diphosphate (ADP), and serotonin (5-hydroxytryptamine [5-HT]) were
purchased from Sigma (St Louis, MO).
Arachidonoyl-trifluoromethyl-ketone (ATFMK) and
N-(4-hydroxyphenyl) arachidonoylamide (AM404) were from
Research Biochemicals International (Natick, MA).
Ethyleneglycol-bis(
