Proc. Natl. Acad. Sci. USA
Vol. 95, pp. 4810–4815, April 1998
Chemistry
Chemical requirements for inhibition of gap junction
communication by the biologically active lipid oleamide
DALE L. BOGER*†, JEAN E. PATTERSON*, XIAOJUN GUAN‡, BENJAMIN F. CRAVATT*, RICHARD A. LERNER*,
AND NORTON B. GILULA‡
Departments of *Chemistry and ‡Cell Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, CA 92037
Communicated by K. Barry Sharpless, The Scripps Research Institute, La Jolla, CA, February 18, 1998 (received for review November 3, 1997)
In addition to its sleep-inducing properties in animals,
oleamide has been shown to effect serotonergic systems (7,
12–14) and to block gap junction communication (15) in a
structurally specific manner. Although it was found to inhibit
the gap junction-mediated chemical and electrical transmission in rat glial cells, it had no inhibitory effect on mechanically
stimulated or glutamate-induced calcium wave propagation,
thereby decoupling two previously indistinguishable glial cell
communication pathways (15). Given the central role of
intercellular chemical and electrical signaling in the central
nervous system, the oleamide inactivation of glial cell gap
junction channels may influence higher order neuronal function including sleep. Herein, we describe a study of the
structural features of oleamide required for this inhibition of
glial cell gap junction communication.
ABSTRACT
Oleamide is an endogenous fatty acid primary amide that possesses sleep-inducing properties in animals and has been shown to effect serotonergic systems and
block gap junction communication in a structurally specific
manner. Herein, the structural features of oleamide required
for inhibition of the gap junction-mediated chemical and
electrical transmission in rat glial cells are defined. The
effective inhibitors fall into two classes of fatty acid primary
amides of which oleamide and arachidonamide are the prototypical members. Of these two, oleamide constitutes the
most effective, and its structural requirements for inhibition
of the gap junction are well defined. It requires a chain length
of 16–24 carbons of which 16–18 carbons appears optimal, a
polarized terminal carbonyl group capable of accepting but
not necessarily donating a hydrogen bond, a D9 cis double
bond, and a hydrophobic methyl terminus. Within these
constraints, a range of modifications are possible, many of
which may be expected to improve in vivo properties. A select
set of agents has been identified that serves both as oleamide
agonists and as inhibitors of fatty acid amide hydrolase, which
is responsible for the rapid inactivation of oleamide.
MATERIALS AND METHODS
Materials. The agents examined were purchased (Sigma,
Pfaltz & Bauer, and Aldrich), prepared as described (3, 4,
16–19), or synthesized following protocols previously detailed
(3, 4, 16–18).
Methods. Rat glial cells (20) were cultured in plastic tissue
culture ware in Richter’s improved minimal essential medium
(Irvine Scientific), supplemented with 10% fetal calf serum
and 50 mgyml gentamicin sulfate, and incubated in a humidified atmosphere of 95% airy5% CO2 at 37°C. The cells were
passaged by trypsinization and used at passages 6–10 and
experimented at '90% confluent. The tested compounds
were dissolved in EtOH and added to the culture media (1
mLyml medium) providing the indicated concentrations (100,
50, or 20 mM) from 1,000-fold higher stock solutions. Control
cultures were treated with EtOH (1 mLyml medium, final 0.1%
EtOH concentration). Microinjection of fluorescent Lucifer
yellow CH dye into cultured cells and quantitation of the dye
transfer frequency to directly adjacent neighboring cells were
performed as described (21). In brief, micropipettes (approximate tip diameter of 0.5 mm) were loaded with the dye
solution (5% Lucifer yellow CH dye in aqueous 0.1 M LiCl) by
backfilling. Cells were visualized by a Nickon Diaphot inverted
phase contrastyepifluorescent microscope and impaled with
dye-filled micropipettes by using an Eppendorf micromanipulator (model 5240). Impaled cells were loaded with dye by
iontophoresis for 2–3 s at 20 nA. After 5 min, the transfer of
dye to directly adjacent cells was determined by using epifluorescent illumination. For each treatment, compound was
maintained in the culture dish for 4 h, and then 10 cells were
microinjected in each of two dishes and the total numbers of
dye-coupled and nondye-coupled adjacent neighboring cells
were calculated and compared with control (0% inhibition) to
determine the percentage of inhibition of dye-coupling.
Oleamide (1) is an endogenous fatty acid primary amide shown
to accumulate in the cerebrospinal fluid under conditions of
sleep deprivation (1–3). In a structurally specific manner, it has
been shown to induce physiological sleep in animals when
administered by i.p. or i.v. injection (1). Consistent with its role
as a prototypical member of a new class of biological signaling
molecules, enzymatic regulation of the endogenous concentrations of oleamide has been described (1, 4–7) or proposed
(8). Fatty acid amide hydrolase (FAAH, oleamide hydrolase)
is an integral membrane protein that degrades 1 to oleic acid,
and potent inhibitors (Ki 5 13 mM–1 nM) of the enzyme have
been detailed (4). The characterization and neuronal distribution of FAAH have been disclosed (5–7), and it was found
to possess the capabilities of hydrolyzing a range of fatty acid
amides including anandamide, which serves as an endogenous
ligand for cannabinoid receptors (9–11). In contrast to anandamide, an appealing feature of the members of this new class
of biological signaling agents is the primary amide, suggesting
that their storage and release may be controlled in a manner
analogous to that of short peptide hormones and messengers
terminating in a primary amide (8).
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviation: FAAH, fatty acid amide hydrolase.
†To whom reprint requests should be addressed. e-mail: boger@
scripps.edu.
© 1998 by The National Academy of Sciences 0027-8424y98y954810-6$2.00y0
PNAS is available online at http:yywww.pnas.org.
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Chemistry: Boger et al.
Proc. Natl. Acad. Sci. USA 95 (1998)
4811
RESULTS
Fatty Acid Primary Amides. The first series examined were
the primary amides (1, 3, 22–24) of the naturally occurring
fatty acids and related synthetic analogs (Table 1). In these
studies, the number, position, and stereochemistry of the
double bond(s) and the length of the agent were assessed.
From these studies, which were assisted with the inclusion of
the primary amides of nonnaturally occurring fatty acids, a
clear depiction of the structural requirements of the endogenous agent emerged. The effective inhibitors of the gap
junction-mediated dye transfer fall into two classes of which
oleamide and arachidonamide (9–11, 25–32) are the prototypical members.
For agents related to oleamide that contain one double
bond, the presence, position, and stereochemistry of the olefin
as well as the chain length were found to have a defined
relationship. The monounsaturated fatty acid primary amides
of 16–24 carbons containing a D9 cis double bond were all
found to be effective inhibitors of the gap junction-mediated
dye transfer. Removal of the D9 double bond (18:0) or its
replacement with a trans double bond (18:19-trans) resulted in
no observable inhibition at the concentrations examined.
Shortening the chain length to 14 carbons (14:19) resulted in
the loss of activity, and lengthening the chain slightly diminished the inhibitory effectiveness. No further substantial trend
in the length was observed with the longer fatty acid primary
amides provided they possessed a D9 cis double bond: 14:19 ,,
16:19 5 18:19 . 20:19 5 22:19 5 24:19. The position of the cis
double bond had a well defined effect on the potency. A D9 cis
olefin exhibited the strongest inhibitory effect, and the potency
typically declined as its position was moved in either direction.
This effect was apparent in the comparisons made in the 18:1
series and was especially clear in the 20:1 series where the
potency declined as the distance from the D9 position increased. Thus, although 18:18 was less effective while 18:17 and
18:16 were as effective as oleamide, the potency decline as the
distance from the D9 position increased was more apparent in
the 20:1 series. Although this location in oleamide is central to
its structure and potentially represents a relationship with
either the carboxamide or methyl terminus, the potent activity
of palmitoleamide (16:19) suggested that it is the positional
relationship with the amide that is important. This relationship
was confirmed in the 20:1, 22:1, and 24:1 series where inhibitory activity was observed with the synthetic D9 olefins rather
than the naturally occurring isomers bearing a cis double bond
9 carbons from the methyl terminus: 20:19 5 20:111, 22:19 .
22:113, and 24:19 . 24:115. In this context, it is interesting that
erucamide (22:113), which has been detected in human cerebrospinal fluid (1), is not an inhibitor of the gap junction and
does not induce physiological sleep in rats.
With the polyunsaturated fatty acid primary amides, those
containing two double bonds exhibited modest activity. Typically, this activity was considerably diminished relative to
oleamide except for linoleamide (18:29,12), which also possesses the cis D9 double bond. Even with linoleamide, the
inhibition potency was lower than oleamide, indicating that the
additional D12 cis double bond was not beneficial. The fatty
acid primary amides containing three double bonds were even
less active with essentially all members being ineffective at 50
mM, including a-linolenamide (20:39,12,15), which incorporates
the D9 cis double bond of oleamide, and 20:38,11,14, which
incorporates an alternative D8 or D11 cis double bond. The
exception to this generalization is g-linolenamide (18:36,9,12),
which proved to be an effective but less potent inhibitor.
Similarly, arachidonamide containing four cis double bonds
was an effective inhibitor of the gap junction-mediated dye
transfer although it was not quite as potent as oleamide. This
effect also was observed with a fatty acid primary amide
containing five but not six cis double bonds.
4812
Chemistry: Boger et al.
These results are consistent with two classes of fatty acid
primary amides that inhibit the gap junction-mediated dye
transfer. The prototypical member of the more potent class is
oleamide, and the second, slightly less effective, class contains
arachidonamide.
The Carboxamide. A study of the requirements at the
carboxylate terminus proved revealing in that it was surprisingly tolerant (Table 2). Oleic acid was found to be inactive at
the concentrations examined, and oleyl alcohol, methyl or
ethyl oleate, and oleyl acetate exhibited progressively reduced
effectiveness. Oleic acid (33, 34), arachidonic acid (35, 36), and
a-linolenic acid (37) have been reported to inhibit the gap
junction in rat cardiac myocytes, striatal astrocytes, and rat
liver epithelial cells, respectively. The limited studies of their
structural requirements for gap junction inhibition including
the chain length, degree and site of unsaturation, and the
stereochemistry of the double bond(s) roughly follow those
Proc. Natl. Acad. Sci. USA 95 (1998)
detailed herein although fewer fatty acids have been examined
in these efforts (33–39). The comparisons detailed in Table 2
suggest that the most effective derivatives of the fatty acids in
rat glial cells are the primary amides. However, a wide range
of secondary or tertiary amides were found to be potent
inhibitors of the gap junction-mediated dye transfer. Thus, the
hydrogen bond donor capability of the primary amides is not
essential to the expression of their properties. Many of these
were equipotent with oleamide, and the series examined
exhibited a clear trend favoring the smaller secondary or
tertiary amide substituents. This trend is evident upon comparing the effects of R 5 NH2 $ MeNH . Et2N .
c-(CH2)4N . PhNH. Although the secondary amides typically
were more potent than the corresponding tertiary amides, the
comparable potency of the N-methyl and N,N-dimethyl amide
(MeNH 5 Me2N) is notable. This potency is consistent with a
requirement for small amide substituents, and the most potent
of the agents examined was the O-methyl,N-methyl hydroxamide (R 5 MeONMe). Although the esters of oleic acid were
inactive at 50–100 mM, oleyl aldehyde proved to be surprisingly
effective while its corresponding dimethyl acetal was inactive.
Oleyl aldehyde was found to be nearly equipotent with oleamide, and the related agent R 5 CF3 possessing a strongly
electrophilic and polarized carbonyl was significantly more
potent than oleamide. In contrast, related but less electrophilic
ketones (R 5 BrCH2 . ClCH2) were substantially less effective. Thus, a smooth trend favoring the more polarized or
electrophilic carbonyls was observed. The behavior of the
former two agents (R 5 H, CF3) is especially significant
because they are also potent inhibitors (Ki 5 190 and 1 nM,
respectively) of fatty acid amide hydrolase (4). Such agents
possess dual activities serving both as effective agonists of
oleamide and as potent inhibitors of the enzyme responsible
for its degradation. Both exhibit a reversible effect on the gap
junction inhibition and removal of the media containing the
agents and replacement with fresh media restored full gap
junction function. Although such inhibitors may exert their
effects by inhibiting FAAH and potentiating the effect of
endogenous oleamide, both the very low levels of FAAH and
endogenous oleamide in rat glial cells are not sufficient to
account for the gap junction inhibition effect.
Thus, a range of effective although sterically undemanding
substitutions can be made for the carboxamide. To date, the
effective inhibitors possess a polarized carbonyl at the C
terminus potentially capable of accepting but not necessarily
donating a hydrogen bond.
Oleyl Ethanolamide, Anandamide, and Related Structures.
An important subset of modified carboxamides is the ethanolamide derivatives (40–43). In our earlier study, anandamide
as well as oleamide were shown to selectively inhibit gap
junction communication (dye transfer and electrical coupling)
without affecting calcium wave transmission although oleamide was more potent (15). This effect in glial cells is distinct
from the report that anandamide and oleyl ethanolamide not
only inhibit gap junction-mediated dye transfer but also block
the propagation of astrocyte calcium waves generated either by
mechanical stimulation or local glutamate application (32).
Consequently, the corresponding ethanolamide of oleic acid
(3) and the bis-(ethanol)amides of oleic and arachidonic acid
were examined (Table 3). Oleamide proved more potent than
oleyl ethanolamide and discernibly more potent than anandamide at 20 mM although both were effective inhibitors at 50
mM. Although the effective properties of the oleyl ethanolamide (R 5 HOCH2CH2NH) may suggest that it represents an
important member of another class of active endogenous
agents, it is less potent than oleamide, N-ethyl oleamide, which
lacks the hydroxyl group, as well as N-propyl oleamide, which
contains a methyl group in place of the hydroxyl group (Table
2). In addition, oleyl propanolamide exhibited comparable
properties. Thus, the hydroxyl group of oleyl ethanolamide
Chemistry: Boger et al.
does not appear to contribute to its gap junction inhibition
properties and actually may diminish them. Similar observations were made in the comparison of arachidonyl amide (R 5
NH2) and anandamide (R 5 HOCH2CH2NH), where the
latter was less potent. Comparable observations have been
made in the examination of anandamide analogs (R 5
CH3CH2CH2NH . HOCH2CH2NH) with their binding to the
central nervous system cannabinoid receptor (25–31). In contrast, the bis-(ethanol)amides were ineffective at inhibiting the
gap junction at 50 mM, which is consistent with their behavior
as bulky tertiary amides that exhibit lower activity.
Recent studies have identified 2-arachidonyl glycerol as a
potential endogenous cannabinoid receptor agonist (44), and
gap junction inhibition by diacylglycerols including 1-oleoyl2-acetylglycerol in Chinese hamster V79 cells has been disclosed (38). Consequently, both racemic and (2S)-1-oleoyl-2acetylglycerol were evaluated as potential inhibitors. Consistent with the behavior of simple oleic acid esters (Table 2),
both were not inhibitors of rat glial gap junction-mediated dye
transfer.
Methyl Terminus. From the studies of the fatty acid primary
amides, the length of the fatty acid chain did not reduce
substantially the gap junction inhibition properties provided it
incorporated the key D9 cis double bond: 18:19 . 20:19 5 22:19
5 24.19. This result indicates that the methyl terminus can
accommodate extensions by a alkyl chain without losing the
agonist properties. Similarly, capping the methyl terminus with
a methyl ether (R 5 CH2OCH3) did not affect the gap junction
inhibition. In contrast, the introduction of polar functionality
at this site eliminated the inhibitory properties. Thus, the
symmetrical 18:19 dicarboxamide as well as the terminal
alcohol and carboxylic acid exhibited no gap junction inhibition at 50 mM (Table 4).
Putative Precursors. The potential that oleamide may be
stored as a N-oleoyl glycinamide derivative whose formation
from oleoyl-CoA is catalyzed by acyl-CoA:glycine Nacyltransferase and released upon a-hydroxylation of glycine
by peptidylglycine a-amidating monooxygenase or a related
enzyme led us to examine a set of N-oleoyl glycine derivatives
even before its potential was demonstrated experimentally (8).
Both N-oleoyl glycine as well as N-oleoyl glycinamide were
ineffective as inhibitors of gap junction-mediated dye transfer
(Table 5). Thus, a putative storage precursor to oleamide as
Proc. Natl. Acad. Sci. USA 95 (1998)
4813
well as N-oleoyl glycinamide, which is representative of a
peptide or protein terminating with an oleoyl glycinamide,
failed to inhibit the gap junction consistent with their role as
inactive precursors (45). N-Oleoyl glycine ethyl ester was an
effective, albeit less potent, inhibitor and likely represents
simply another allowable N-substitution on the terminal carboxamide (cf. Table 2). In contrast, N-oleoyl sarcosine as well
as its ethyl ester were found to be effective inhibitors. Initially
surprising, this observation suggests that the intramolecular
hydrogen-bonded rotamer of the free acid or the deprotonated
carboxylate, which would be observed with N-oleoyl glycine
but less favored with N-oleoyl sarcosine, ties up the amide
carbonyl disrupting its intermolecular hydrogen bond acceptor
capabilities that may be required for gap junction inhibition.
Modifications in the Olefin. A series of agents (2–10) was
examined to define the role of the olefin and to provide insights
into a conformational dependence on activity (Table 6). Thus,
whereas the saturated agent octadecanamide and the trans-9octadecenamide were ineffective (cf. Table 1), 9-octadecynamide (2) was a potent inhibitor, being only slightly less active
than oleamide. Its conformational characteristics, which more
closely resemble those of the inactive 18:0 and 18:19-trans than
oleamide, suggest that the appropriate presentation of a
p-system in addition to the conformation of the cis olefin may
be important. Consistent with this, the epoxide 3 was inactive
and the cyclopropane 4 was less active although both may
closely mimic the conformational characteristics of oleamide.
Although there are a number of plausible explanations for this,
4 may benefit from the partial p characteristics of the cyclopropane. Alternatively, the incorporation of the epoxide oxygen in 3 may alter the hydrophobic character of the agent and
4814
Chemistry: Boger et al.
diminish gap junction inhibition. Consistent with either explanation, both cis- and trans-5 and -6 proved to be weak inhibitors
(cis . trans) of the gap junction-mediated dye transfer, and 5,
which possesses the cis D9 double of oleamide, was more
potent. The removal of the double bond reduced but did not
eliminate the inhibition properties, suggesting an important
role for both the p-character of the double bond as well as its
imposed conformational preference for a hairpin conformation. Consistent with this, incorporation of a benzene ring into
the structure provided the agents 7–10 that inhibit the gap
junction-mediated dye transfer. The most potent in the series
was 8, being nearly equipotent with oleamide, and it most
closely mimics the hairpin conformation of oleamide. This
potency was followed by that of 7, which represents an
extended version of oleamide. Both 9 and 10 were less effective, and both mimic alternatives to the hairpin conformation
of oleamide.
Modifications in the Linking Chain and Enzyme Inhibitors:
Dual Activities. In the examination of the fatty acid primary
amides, it was established that increasing or shortening the
distance between the carboxamide and cis double bond generally diminished the gap junction inhibition. Additional modifications in the 7 carbon chain linking the olefin and carboxamide also were examined (Table 7). The first set of agents
contain modifications that slow the rate of degradation by
enzymatic hydrolysis (1, 4, 12–14, 46). Both a-methyl oleamide
Proc. Natl. Acad. Sci. USA 95 (1998)
and a,a-dimethyl oleamide were potent inhibitors of the gap
junction, and both have been shown to be subject to slower
FAAH hydrolysis than oleamide (12–14). In addition, the
agents containing a terminal primary carbamate (X 5 O) or
urethane (X 5 NH) were also potent inhibitors. The latter is
especially interesting because the corresponding urethane of
oleyl amine, which is two carbons longer in length and whose
double bond resides not 9 but 11 carbons from the terminal
carbonyl, is essentially inactive (Table 2). Similarly, a-hydroxy
oleamide (X 5 CHOH), a-chlorooleamide (X 5 CHCl), and
a-acetylthiooleamide (X 5 CHSAc) were effective inhibitors,
although the free thiol (X 5 CHSH) was ineffective. This latter
agent suffers from disulfide formation that may account for its
ineffectiveness. The last two agents in Table 7 constitute
analogs that are effective (Ki 5 16 and 17 nM, respectively)
inhibitors of FAAH (4). Both serve as oleamide agonists and
as inhibitors (4, 47) of FAAH responsible for its degradation.
For both, the gap junction inhibition was reversible and
restored to full activity after suspension of the glial cells in
fresh media.
DISCUSSION
The disclosure that oleamide inhibits the gap junctionmediated intercellular chemical and electrical transmission
without effecting calcium wave propagation in rat glial cells
(15) suggests that this may constitute an important site of
action for this new class of biologically active lipids. The
effective inhibitors of the gap junction fall into two classes of
fatty acid primary amides of which oleamide and arachidonamide are the prototypical members. Of these, oleamide
constitutes the most effective and its structural requirements
for inhibition of the gap junction are well defined. The
inhibition requires a chain length of 16–24 carbons of which
16–18 appears optimal, a polarized terminal carbonyl capable
of accepting but not necessarily donating a hydrogen bond, a
D9 cis double bond, and a hydrophobic methyl terminus.
Within these constraints, a range of modifications are acceptable, many of which may be used to enhance in vivo properties.
In addition to those that would slow or avoid inactivation by
FAAH hydrolysis (5), a set of especially interesting agents have
been identified that serve both as oleamide agonists and as
FAAH inhibitors (4).
The inhibition of the gap junction by oleamide is consistent
with a number of mechanisms (48). Perturbations in the bulk
Chemistry: Boger et al.
membrane fluidity or the membrane–protein interface that
would effect the conformation of the membrane-bound proteins as well as direct interaction with the gap junction proteins
have been discussed (15, 48). Similarly, both progressive
closure and ultimately collapse of the gap junction as well as
gated closure of the gap junction have been considered. The
compounds disclosed herein and the defined structural features of oleamide responsible for the properties may help
distinguish among the mechanisms as well as provide new
neuromodulatory agents applicable to sleep or mood disorders, analgesia, and disorders associated with higher neuronal
function.
We gratefully acknowledge the Skaggs Institute for Chemical Biology, the National Institutes of Health (CA42056, D.L.B.), and the
National Science Foundation (predoctoral fellowship to B.F.C.) for
financial support. We thank Dr. J. E. Trosko for providing the rat glial
cells and Dr. H. J. Waller for assembling the iontophoresis injector
used for this study.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Cravatt, B. F., Prospero-Garcia, O., Siuzdak, G., Gilula, N. B.,
Henriksen, S. J., Boger, D. L. & Lerner, R. A. (1995) Science 268,
1506–1509.
Lerner, R. A., Siuzdak, G., Prospero-Garcia, O., Henriksen, S. J.,
Boger, D. L. & Cravatt, B. F. (1994) Proc. Natl. Acad. Sci. USA
91, 9505–9508.
Cravatt, B. F., Lerner, R. A. & Boger, D. L. (1996) J. Am. Chem.
Soc. 118, 580–590.
Patterson, J. E., Ollmann, I. R., Cravatt, B. F., Boger, D. L.,
Wong, C.-H. & Lerner, R. A. (1996) J. Am. Chem. Soc. 118,
5938–5945.
Cravatt, B. F., Giang, D. K., Mayfield, S. P., Boger, D. L., Lerner,
R. A. & Gilula, N. B. (1996) Nature (London) 384, 83–87.
Giang, D. K. & Cravatt, B. F. (1997) Proc. Natl. Acad. Sci. USA
94, 2238–2242.
Thomas, E. A., Cravatt, B. F., Danielson, P. E., Gilula, N. B. &
Sutcliffe, G. (1997) J. Neurosci. Res. 50, 1047–1052.
Merkler, D. J., Merkler, K. A., Stern, W. & Fleming, F. F. (1996)
Arch. Biochem. Biophys. 330, 430–434.
Devane, W. A., Hanus, L., Breuer, A., Pertwee, R. G., Stevenson,
L. A., Griffin, G., Gibson, D., Mandelbaum, A., Etinger, A. &
Mechoulam, R. (1992) Science 258, 1946–1949.
Johnson, D. E., Heald, S. L., Dally, R. D. & Janis, R. A. (1993)
Prostaglandins Leukotrienes Essent. Fatty Acids 48, 429–437.
Di Marzo, V. & Fontana, A. (1995) Prostaglandins Leukotrienes
Essent. Fatty Acids 53, 1–11.
Huidobro-Toro, J. P. & Harris, R. A. (1996) Proc. Natl. Acad. Sci.
USA 93, 8078–8082.
Thomas, E. A., Carson, M. J., Neal, M. J. & Sutcliff, J. G. (1997)
Proc. Natl. Acad. Sci. USA 94, 14115–14119.
Boger, D. L., Patterson, J. E. & Jin, Q. (1998) Proc. Natl. Acad.
Sci. USA 95, 4102–4107.
Guan, X., Cravatt, B. F., Ehring, G. R., Hall, J. E., Boger, D. L.,
Lerner, R. A. & Gilula, N. B. (1997) J. Cell Biol. 139, 1785–1792.
Roe, E. T., Scanlan, J. T. & Swern, D. (1949) J. Am. Chem. Soc.
71, 2215–2218.
Roe, E. T., Miles, T. D. & Swern, D. (1952) J. Am. Chem. Soc.
74, 3442–3443.
Simmons, H. E. & Smith, R. D. (1959) J. Am. Chem. Soc. 81,
4256–4264.
Patterson, J. E. (1997) Ph.D. thesis (The Scripps Research
Institute, La Jolla, CA).
Proc. Natl. Acad. Sci. USA 95 (1998)
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
4815
Suter, S., Trosko, J. E., El-Fouly, M. H., Lockwood, L. R. &
Koestner, A. (1987) Fundam. Appl. Toxicol. 9, 785–794.
Guan, X. J., Bonney, W. J. & Ruch, R. J. (1995) Toxicol. Appl.
Pharmacol. 130, 79–86.
Arafat, E. S., Trimble, J. W., Andersen, R. N., Dass, C. &
Desiderio, D. M. (1989) Life Sci. 45, 1679–1687.
Wakamatsu, K., Masaki, T., Itoh, F., Kondo, K. & Katsuichi, S.
(1990) Biochem. Biophys. Res. Commun. 168, 423–429.
Jain, M. K., Ghomashchi, F., Yu, B.-Z., Bayburt, T., Murphy, D.,
Houck, D., Brownell, J., Reid, J. C., Solowiej, J. E., Wong, S.-M.,
et al. (1992) J. Med. Chem. 35, 3584–3586.
Pinto, J. C., Potie, F., Rice, K. C., Boring, D., Johnson, M. R.,
Evans, D. M., Wilken, G. H., Cantrell, C. H. & Howlett, A. C.
(1994) Mol. Pharmacol. 46, 516–522.
Felder, C. C., Briley, E. M., Axelrod, J., Simpson, J. T., Mackie,
K. & Devane, W. A. (1993) Proc. Natl. Acad. Sci. USA 90,
7656–7660.
Abadji, V., Lin, S., Taha, G., Griffin, G., Stevenson, L. A.,
Pertwee, R. G. & Makriyannis, A. (1994) J. Med. Chem. 37,
1889–1893.
Adams, I. B., Ryan, W., Singer, M., Thomas, B. F., Compton,
D. R., Razdan, R. K. & Martin, B. R. (1995) J. Pharmacol. Exp.
Ther. 273, 1172–1181.
Adams, I. B., Ryan, W., Singer, M., Razdan, R. K., Compton,
D. R. & Martin, B. R. (1995) Life Sci. 56, 2041–2048.
Priller, J., Briley, E. M., Mansouri, J., Devane, W. A., Mackie, K.
& Felder, C. C. (1995) Mol. Pharmacol. 48, 288–292.
Khanolkar, A. D., Abadji, V., Lin, S., Hill, W. A. G., Taha, G.,
Abouzid, K., Meng, Z., Fan, P. & Makriyannis, A. (1996) J. Med.
Chem. 39, 4515–4519.
Venance, L., Piomelli, D., Glowinski, J. & Glaume, C. (1995)
Nature (London) 376, 590–594.
Hirschi, K. K., Minnich, B. N., Moore, L. K. & Burt, J. M. (1993)
Am. J. Physiol. 265, C1517–C1526.
Burt, J. M., Massey, K. D. & Minnich, B. N. (1991) Am. J. Physiol.
260, C439–C448.
Giaume, C., Marin, P., Cordier, J., Glowinski, J. & Premont, J.
(1991) Proc. Natl. Acad. Sci. USA 88, 5577–5581.
Massey, K. D., Minnich, B. N. & Burt, J. M. (1992) Am. J. Physiol.
263, C494–C501.
Hasler, C. M., Bennink, M. R. & Trosko, J. E. (1991) Am. J.
Physiol. 261, C161–C168.
Aylsworth, C. F., Trosko, J. E. & Welsch, C. W. (1986) Cancer
Res. 46, 4527–4533.
Spray, D. C. & Burt, J. M. (1990) Am. J. Physiol. 258, C195–C205.
Bachur, N. R., Masek, K., Melmon, K. L. & Udenfriend, S. (1965)
J. Biol. Chem. 240, 1019–1024.
Ramachandran, C. K., Murray, D. K. & Nelson, D. H. (1992)
Biochem. Arch. 8, 369–377.
Schmid, H. H. O., Schmid, P. C. & Natarajan, V. (1990) Prog.
Lipid Res. 29, 1–43.
Hanus, L., Gopher, A., Almog, S. & Mechoulam, R. (1993)
J. Med. Chem. 36, 3032–3034.
Mechoulam, R., Ben-Shabat, S., Hanus, L., Ligumsky, M., Kaminski, N. E., Schatz, A. R., Gopher, A., Almog, S., Martin, B. R.,
Compton, D. R., et al. (1995) Biochem. Pharmacol. 50, 83–90.
Matsumoto, M., Park, J., Sugano, K. & Yamada, T. (1987) Am. J.
Physiol. 252, G315–G319.
Maurelli, S., Bisogno, T., De Petrocellis, L., Di Luccia, A.,
Marino, G. & Di Marzo, V. (1995) FEBS Lett. 377, 82–86.
Koutek, B., Prestwich, G. D., Howlett, A. C., Chin, S. A.,
Salehani, D., Akhavan, N. & Deutsch, D. G. (1994) J. Biol. Chem.
269, 22937–22940.
Lerner, R. A. (1997) Proc. Natl. Acad. Sci. USA 94, 13375–13377.