N-arachidonoyl L-, an endocannabinoid-like brain constituent with vasodilatory properties

Garry Milman*†, Yehoshua Maor*†, Saleh Abu-Lafi‡, Michal Horowitz§, Ruth Gallily¶, Sandor Batkaiʈ, Fong-Ming Moʈ, Laszlo Offertalerʈ, Pal Pacherʈ, George Kunosʈ**, and Raphael Mechoulam*,**

Departments of *Medicinal Chemistry and Natural Products and ¶Immunology, Medical Faculty, and §Laboratory of Environmental Physiology, Faculty of Dental Medicine, Hebrew University, Jerusalem 91120, Israel; ‡Chemistry and Chemical Technology Department, Al-Quds University, Abu-Deis, Palestinian Authority; and ʈLaboratory of Physiologic Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892

Communicated by L. L. Iversen, University of Oxford, Oxford, United Kingdom, December 19, 2005 (received for review September 22, 2005) The endocannabinoid N-arachidonoyl (anandam- Results ide), found both in the CNS and in the periphery, plays a role in ARA-S was extracted from bovine brains by the procedure numerous physiological systems. One might expect that the chem- described below to yield extract A. Two routes were followed to ically related N-arachidonoyl-L-serine (ARA-S) could also be formed identify ARA-S. In the first, extract A was silylated with alongside . We have now isolated ARA-S from bovine N,O-bis(trimethyl-silyl)trifluoroacetamide and analyzed by GC- brain and elucidated its structure by comparison with synthetic MS. The peak of synthetic di-trimethyl-silyl (di-TMS)-ARA-S ARA-S. Contrary to anandamide, ARA-S binds very weakly to appears at 17.32 min. The mass spectra of the peak from silylated CB1 and CB2 or vanilloid TRPV1 (transient synthetic material and of extract A, eluting at a comparable time potential vanilloid 1) receptors. However, it produces endotheli- period, were identical. um-dependent of rat isolated mesenteric arteries and The high-resolution mass spectra of endogenous and synthetic abdominal aorta and stimulates phosphorylation of p44͞42 mito- ARA-S (as their di-TMS derivatives) were also identical (Fig. 1B). gen-activated protein (MAP) kinase and protein kinase B͞Akt in Both natural di-TMS-ARA-S and synthetic di-TMS-ARA-S eluted cultured endothelial cells. ARA-S also suppresses LPS-induced for- after Ϸ18 min. Natural di-TMS-ARA-S was found to have a mation of TNF-␣ in a murine macrophage cell line and in wild-type molecular weight ion at 535.3501 m͞z, which is within 2.3 ppm of ͞ mice, as well as in mice deficient in CB1 or CB2 receptors. Many of the calculated mass (535.3513 m z) and indicates an elemental these effects parallel those reported for abnormal composition of C29H53NO4Si2, which is consistent with the di-TMS (Abn-CBD), a synthetic of a putative novel cannabinoid- derivative of the proposed structure (Fig. 1A). ARA-S is therefore type receptor. Hence, ARA-S may represent an endogenous agonist aC23H37NO4 compound, with a molecular weight of 391.5. Ϫ for this receptor. ARA-S is not stable at room temperature or at 20°C (without a solvent). However, kept at Ϫ20°C in ethanol it was stable for at ͉ anandamide ͉ ͉ endothelium ͉ least up to 2 months. reactive oxygen intermediates The second route involved methylation of extract A, followed by silylation. We compared the mass spectrum of the TMS derivative of the compound peak, eluting at 15.87 min, with that of the of he identification, structural elucidation, and syntheses of the synthetic TMS-ARA-S methyl ester, which elutes at the same time. Tplant cannabinoids in the early 1960s led to thorough investi- The mass spectra were identical, with molecular ion [M]ϩ 477 m͞z. gations of the chemistry, metabolism, and pharmacology of these The NMR spectral data of synthetic ARA-S methyl ester are compounds, in particular of the psychoactive constituent ⌬9- presented in Supporting Text, which is published as supporting (1, 2). However, until the late 1980s and early information on the PNAS web site. 1990s, when specific receptors were identified and shortly thereafter The stereochemistry of ARA-S was established by comparison of cloned, the mechanism of the numerous cannabinoid actions re- the elution time of natural ARA-S methyl ester with the synthetic mained an enigma (3–5). Two main receptors are now known: the L and D methyl esters of ARA-S on a HPLC chiral column. Overlaid CB1 receptor, found in the CNS, as well as in some peripheral chromatograms of synthetic arachidonoyl-D-serine or ARA-S tissues, and the CB2 receptor, found predominantly in the immune methyl esters with methylated ARA-S from brain-derived material system (6–8). Additional, not yet fully identified receptors are showed optical identity of the methyl ester of the natural material present both in the CNS and in the periphery (6, 9, 10). with the synthetic L-enantiomer (elution time 26.7 min) and non- Because receptors in mammals are not formed to encounter a identity with the synthetic D-enantiomer (elution time 18.3 min) plant constituent, research was initiated to discover endogenous (Fig. 1C). ligands. In the 1990s two endogenous cannabinoids (endocannabi- Contrary to anandamide, ARA-S binds very weakly to CB1 Ͼ noids) were identified, N-arachidonoyl ethanolamine (anandam- receptors in mouse cerebellar membranes (Ki 10,000 nM). ide) (11) and 2-arachidonoyl-glycerol (12, 13). Additional endo- Furthermore, at concentrations up to and including 30 ␮M, ARA-S cannabinoids have been reported, but their biological roles are yet failed to displace radioligand binding to rat CB2 receptors or rat obscure (6, 14). Anandamide and 2-arachidonoyl-glycerol have TRPV1 (transient receptor potential vanilloid 1) receptors. large spectrum of physiological actions, most of which are associ- ARA-S relaxes rat isolated mesenteric arteries (EC50, 550 nM) Ϸ ated with the neural and immune systems. However, cardiovascular and abdominal aorta (EC50, 1,200 nM). These effects resemble effects, which are in part CB1-mediated (15), are also well estab- lished (9, 14, 16). Conflict of interest statement: No conflicts declared. Anandamide is a product of (17). Abbreviations: CBD, cannabidiol; Abn-CBD, abnormal CBD; ARA-S, N-arachidonoyl-L- Because phosphatidylserine is found alongside phosphatidyleth- serine; HUVEC, human umbilical vein endothelial cell; PTX, pertussis toxin; TMS, trimethyl- anolamine in body tissues, one might expect that arachidonoyl-L- silyl; MAP, mitogen-activated protein. serine (ARA-S) is also an endogenous constituent (see Fig. 1A for †G.M. and Y.M. contributed equally to this work. the structures of anandamide and ARA-S). We report that we have **To whom correspondence may be addressed. E-mail: [email protected] or isolated ARA-S from bovine brain and have evaluated some of its [email protected]. biological properties. © 2006 by The National Academy of Sciences of the USA

2428–2433 ͉ PNAS ͉ February 14, 2006 ͉ vol. 103 ͉ no. 7 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510676103 Downloaded by guest on October 1, 2021 Fig. 1. Identification of ARA-S in bovine brain. (A) Structures of anandamide and ARA-S. (B) Electron impact high-resolution mass spectra of synthetic ARA-S (a) and endogenous ARA-S (b) from bovine brain. Both spectra are after derivatization with N,O-bis(trimethyl-silyl)trifluoroacetamide. (C) Overlay chromatogram of natural and synthetic D-(Ϸ18 min), L-(Ϸ26 min) ARA-S methyl esters obtained with HPLC chiral chromatography.

those of abnormal cannabidiol (Abn-CBD), a synthetic agonist of component in the effect of ARA-S in mesenteric arteries. In these a novel cannabinoid-type receptor (18). In rat aorta, 1 ␮M SR- preparations, Abn-CBD also elicited full vasorelaxation (EC50,2 141716A or SR-144528 did not antagonize ARA-S-induced vasore- ␮M), sensitive to inhibition by O-1918 but not by PTX (data not laxation (data not shown), indicating the lack of involvement of CB1 shown). or CB2 receptors. In the presence of indomethacin (10 ␮M) the Exposure of cultured human umbilical vein endothelial cells ARA-S effects were unchanged. The cannabidiol (CBD) analog (HUVEC) to 1 ␮M ARA-S or 30 ␮M Abn-CBD for 30 min ͞

O-1918 is an inhibitor of the mesenteric vasorelaxant effects of significantly increased the phosphorylation of Akt and p44 42 PHARMACOLOGY Abn-CBD (18, 19) and anandamide (20). O-1918 at 10 ␮M did not mitogen-activated protein (MAP) kinase, which was attenuated by antagonize ARA-S-induced relaxation of aortic rings (Fig. 2A), preincubation with 400 ng͞ml PTX (Fig. 3A). The concentration which was endothelium-dependent: maximum relaxation was 60 Ϯ dependence of these effects is illustrated in Fig. 3B. These findings 4% in intact preparations vs. 29 Ϯ 1% in denuded preparations suggest that a receptor similar to that in rat aorta is present in (Fig. 2A). The aortic relaxant effect of ARA-S was blocked by HUVEC and is involved in mediating the effect of ARA-S on MAP pertussis toxin (PTX; 0.5 ␮g͞ml,Fig.2B), which similarly inhibited kinase and Akt. The PTX sensitivity of the response suggests the aortic relaxation by Abn-CBD (data not shown). involvement of Gi͞Go. The relaxant effect of ARA-S was less endothelium-dependent Treatment of murine RAW264.7 macrophages with ARA-S in mesenteric artery segments than in aorta: denudation right- suppressed the production of zymosan-induced reactive oxygen shifted the ARA-S concentration–response curve without reducing intermediates (ROI). The inhibitory effect was concentration- the maximal response (Fig. 2C). Unlike in aorta, PTX did not dependent and reached 45% at 76.6 ␮M ARA-S. ARA-S also influence the ARA-S response (Fig. 2D). Also, 10 ␮M O-1918 inhibited the LPS-induced increase of NO production by primary significantly inhibited mesenteric relaxation by ARA-S, and the murine macrophages, which reached 50% at the highest concen- inhibition remained significant in endothelium-denuded prepara- tration tested. In the same cells, 24-h incubation with ARA-S tions (Fig. 2C). Thus, the different behavior of the two preparations inhibited TNF-␣ production, reaching 78% inhibition at the highest may be related to the presence of an endothelium-independent concentration of ARA-S used (see Fig. 5, which is published as

Milman et al. PNAS ͉ February 14, 2006 ͉ vol. 103 ͉ no. 7 ͉ 2429 Downloaded by guest on October 1, 2021 Fig. 2. ARA-S-induced vasorelaxation in rat aortic rings (A and B) and mesenteric artery segments (C and D). (A) The aortic relaxant effect of ARA-S (filled squares) is not antagonized by 10 ␮M O-1918 (open squares) and is nearly eliminated by endothelial denu- dation (filled circles). (B) Relaxation of aortic rings by ARA-S (filled squares) is blocked by PTX (open trian- gles). (C) Mesenteric vasorelaxation by ARA-S is inhib- ited by 10 ␮M O-1918 (open symbols) in endothelium- intact (squares) and endothelium-denuded (circles) preparations. (D) Mesenteric vasorelaxation by ARA-S (filled squares) is unaffected by PTX (open triangles). Points and vertical bars are means Ϯ SE from four to six experiments.

supporting information on the PNAS web site). Contrary to known two-step procedure (21). The chromatographic behavior ARA-S, its synthetic D-enantiomer failed to elicit any of these (elution time on GC-MS) and mass spectra (Fig. 1B)ofthe effects (data not shown). natural and synthetic materials (after TMS silylation on both the Treatment of wild-type mice with 10 mg͞kg ARA-S or 20 mg͞kg hydroxyl and carboxyl groups) were identical. The methyl ester Abn-CBD i.p. reduced LPS-induced TNF-␣ production by Ϸ40%, of synthetic and endogenous ARA-S (both in their mono-TMS whereas the potent CB1͞CB2 agonist HU-210 (50 ␮g͞kg) reduced forms) also have identical retention time and mass spectra. plasma TNF-␣ levels by Ϸ80%. These effects remained significant A high-resolution MS measurement of endogenous ARA-S (as Ϫ/Ϫ Ϫ/Ϫ in both CB1 and CB2 mice, although the size of the reduction the di-TMS derivative, Fig. 1B) indicated the elemental composi- Ϫ/Ϫ was lower in CB1 mice than in control mice (Fig. 4). tion C23H37NO4 for ARA-S and molecular weight 391.5 (Fig. 1A). On a HPLC chiral column the elution time of ARA-S methyl Discussion ester was identical to that of synthetic L-enantiomer and different We describe the isolation and structural elucidation of a novel from that of synthetic D-enantiomer of methylated ARA-S (Fig. endocannabinoid-like component, ARA-S, from bovine brain. 1C). Thus, the endogenous isolated from brain is ARA-S. For comparison, this compound was synthesized following a ARA-S binds very weakly to the CB1 receptor with a potency of Ͻ1% of that of anandamide and does not bind to either the CB2 or the TRPV1 receptor. Because anandamide can elicit vasodilation

Fig. 3. Activation of MAP kinase and protein kinase B͞Akt. (A) ARA-S (1 ␮M) and Abn-CBD (30 ␮M) increase the phosphorylation of protein kinase B͞Akt Fig. 4. The effect of ARA-S (10 mg͞kg), Abn-CBD (20 mg͞kg), and HU-210 (50 Ϫ/Ϫ (Top) and p44͞42 MAP kinase (Middle), effects inhibited by PTX (400 ng͞ml). ␮g͞kg) on LPS-induced plasma TNF-␣ levels in wild-type (n ϭ 32), CB1 (n ϭ Ϫ/Ϫ Western blotting is described in Materials and Methods. ␤-Actin (Bottom) was 5), and CB2 (n ϭ 4) mice. Basal levels of TNF-␣ were 30 Ϯ 10, 39 Ϯ 15, and used as loading control. This experiment was replicated two more times with 35 Ϯ 13 pg͞ml, and LPS-stimulated levels were 3,407 Ϯ 197, 3,642 Ϯ 326, and similar results. (B) Concentration-dependent phosphorylation of p44͞42 MAP 6,877 Ϯ 857 pg͞ml in the three groups, respectively. Significant difference kinase by ARA-S and Abn-CBD. from vehicle-treated values is indicated by * (P Ͻ 0.05) and ** (P Ͻ 0.001).

2430 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510676103 Milman et al. Downloaded by guest on October 1, 2021 by a mechanism that does not involve CB1,CB2, or TRPV1 lator peptide CGRP (41). This mechanism may account for the receptors (9, 22, 23), we tested ARA-S for possible vasodilatory endothelium-independent component of vasodilation by anandam- activity. ARA-S is indeed vasorelaxant in an endothelium- ide in some vascular beds, but not in others (9, 40). However, this dependent manner in both rat abdominal aorta and mesenteric effect of anandamide is not sensitive to TRPV1 receptor blockade artery segments. The effect is independent of CB1 and CB2 (28), and neither is the effect of Abn-CBD (9, 18, 19). The effect receptors, as indicated by its resistance to blockade by CB1 or CB2 of ARA-S is similarly independent of the TRPV1 receptor, because antagonists. However, there are also differences between the effect it does not bind to this receptor. of ARA-S in aorta and mesenteric arteries, as discussed below. In summary, the brain produces a substance which, although In intact animals, anandamide elicits hypotension mediated chemically related to anandamide, binds very weakly to the known exclusively by CB1 receptors (9, 24). This effect is due primarily to cannabinoid receptors and parallels the vasodilatory activity of decreased cardiac contractility and output, with little change in synthetic Abn-CBD. Its activity is endothelium-dependent and vascular resistance (25). Although CB1 receptor activation may lead PTX-sensitive in some models but not in others. These results to localized vasodilation in the coronary (26) or cerebral (27) reinforce the hypothesis that the effects of ARA-S may be in part vasculatures, additional receptors distinct from CB1,CB2,or mediated by a Gi͞Go-coupled receptor distinct from CB1 and CB2 TRPV1 have been implicated in anandamide-induced vasodilation and similar to the Abn-CBD-sensitive receptor. (9, 20, 23). In the rat mesenteric vascular bed, anandamide causes Anandamide and 2-arachidonoyl-glycerol have been reported to endothelium-dependent vasodilation inhibited by micromolar con- exert their vasodilatory effect by degradation to centrations of the CB1 antagonist SR141716A and by PTX (9, 28, (42, 43). Vasodilation by ARA-S is not elicited through a similar 29). However, the effect is not inhibited by another CB1 antagonist, mechanism, because it is unaffected by indomethacin, a nonselec- AM251 (20, 29, 30), and potent synthetic CB1͞CB2 do not tive cyclooxygenase inhibitor. elicit mesenteric vasodilation (31). Furthermore, mesenteric vaso- ARA-S suppresses formation of reactive oxygen intermediates, dilation by anandamide remains unchanged in CB1 knockout or NO, and TNF-␣ in a murine macrophage cell line, as well as TNF-␣ CB1͞CB2 double knockout mice (9), which rules out the involve- in vivo, indicating that it may also have antiinflammatory activity. ment of these receptors. Capsazepine fails to antagonize the Interestingly, anandamide and certain N-acyl-vanillamines have endothelium-dependent vasodilator effect of anandamide (9, 32), been reported to inhibit NF-␬B activation through a cannabinoid arguing against TRPV1 receptor involvement. and vanilloid receptor-independent pathway (44). Cannabinoid Abn-CBD, a synthetic isomer of the behaviorally inactive phy- inhibition of TNF-␣ production in vivo also has features incompat- tocannabinoid CBD, elicits endothelium-dependent, capsazepine- ible with the involvement of CB1 or CB2 receptors (45). ARA-S and insensitive mesenteric vasodilation (9, 18), unaffected by CB1 or Abn-CBD maintain their ability to inhibit TNF-␣ production in CB2 antagonists but inhibited by CBD (9) or O-1918 (18), and is mice deficient in CB1 or CB2 receptors, providing strong evidence moderately sensitive to resiniferotoxin (Ϸ2-fold increase in EC50; for an alternative mechanism. Whether the Abn-CBD-sensitive see ref. 18). This finding suggests that Abn-CBD is a selective receptor is the same in macrophages and in vascular endothelium agonist and CBD and O-1918 are selective antagonists of an remains to be determined. The effects of ARA-S in cultured atypical endothelial receptor coupled to Gi͞Go. Ho and Hiley (19) RAW264.7 macrophages require high concentrations of this lipid, confirmed the ability of O-1918 to inhibit the mesenteric vasore- and it has yet to be shown that such endogenous levels of ARA-S laxant effect of Abn-CBD but found it to be PTX-resistant, similar can be reached. Nevertheless, the inability of the D-enantiomer of to the present findings with both ARA-S and Abn-CBD in mes- ARA-S to mimic these effects suggests that they are initiated enteric artery segments. In these preparations, endothelial denu- through a receptor-mediated mechanism. dation caused only a parallel shift of the ARA-S dose–response The above actions of ARA-S suggest that it may be involved in curve (Fig. 2C), whereas in aorta it nearly abolished it (Fig. 2B). the homeostasis of the vascular system in addition to acting as a Furthermore, in mesenteric arteries O-1918 inhibited the effect of vascular protective agent. Cannabinoids have long been known as ARA-S even after removal of the endothelium (Fig. 2C). There- antiinflammatory agents (45, 46), but some of them are psycho- fore, both the inhibition by O-1918 and the resistance to PTX may tropic. ARA-S is devoid of psychotropic activity and may be be properties of the endothelium-independent component of the involved in the pathology of inflammatory conditions including effect of ARA-S and Abn-CBD, which is present in mesenteric atherosclerosis, a state in which altered inflammation, cell migra- arteries but not in aorta. Qualitative differences in receptor expres- tion, and altered vascular elasticity are present. Our results point sion are known to exist between large and small vessels even in the toward the importance of ARA-S as a vascular protective agent. same vascular bed (33). The PTX sensitivity of ARA-S responses in HUVEC is compatible with such a scheme, which suggests that Materials and Methods there may be more than one vascular receptor for these novel Chemicals. Abn-CBD [(Ϫ)-4-(3–3,4-trans-p-menthadien-[1,8]-yl)- ligands. olivetol] was synthesized as described (47). O-1918 was kindly The present findings reveal the similar profile of actions of provided by Raj Razdan (Organix, Inc., Woburn, MA) (18). PHARMACOLOGY ARA-S and Abn-CBD, implicating parallel mechanisms of action. SR-141716A (a CB1 ) and SR-144528 (a CB2 Other endocannabinoids, including N-arachidonoyl (34) receptor antagonist) were from Triangle Research Laboratories. and virodhamine (35), also interact with the Abn-CBD-sensitive ARA-S and arachidonoyl-D-serine were synthesized as reported in endothelial receptor (30, 36), in addition to their actions at CB1 and ref. 21. N,O-bis(trimethyl-silyl)trifluoroacetamide, containing 1% TRPV1 receptors. In contrast, ARA-S does not bind to CB1,CB2, trimethylchlorosilane, phenylephrine HCl, indomethacin, luminol, or TRPV1 receptors, so it is the first selective endogenous of LPS (Escherichia coli 0127:B8), and zymosan were from Sigma. this putative endothelial receptor. In HUVEC, activation of this PTX was obtained from List Biological Laboratories (Campbell, receptor by both ARA-S and Abn-CBD results in phosphorylation CA) and from Sigma. Antibodies against the phosphorylated and of p44͞42 MAP kinase and protein kinase B͞Akt (Fig. 3), pathways unphosphorylated forms of p44͞42 MAP kinase and Akt were involved in endothelial cell migration (37). This finding suggests from New England Biolabs. Low-protein medium was from Bio- that ARA-S may be involved in the regulation of angiogenesis. In logical Industries (Beit Haemek, Israel), and Ficoll Hypaque– microglia, Abn-CBD triggers endothelial cell migration, an impor- Histopaque (density 1.077) was from Sigma. Transwell trays of tant component of neuroinflammation (38). 5-␮m pore were from Corning, ELISA reagents were purchased Relaxation of isolated arteries by anandamide is partly mediated from BD Biosciences (San Diego), and RAW 264.7 cells were by TRPV1 receptors (39–41). Anandamide binds to TRPV1 re- obtained from American Type Culture Collection. DMEM, FCS, ceptors with low affinity (8), resulting in the release of the vasodi- and Hanks’ balanced salt solution (HBSS) were from Biological

Milman et al. PNAS ͉ February 14, 2006 ͉ vol. 103 ͉ no. 7 ͉ 2431 Downloaded by guest on October 1, 2021 Industries (Beit Haemek, Israel), and EGM-2 Bullekit was from purity calculations confirmed that the ARA-S eluted peaks were BioWhittaker. For the in vitro experiments, Abn-CBD, O-1918, and homogeneous; i.e., no coelution was detected. ARA-S were dissolved in ethanol. To determine the optical properties of the isolated natural ARA-S, the methyl esters of arachidonoyl-D-serine and ARA-S Animals. Heterozygote breeding pairs for mice with deleted cb1 or were prepared (see below) and resolved by chiral chromatography. Ϫ/Ϫ Ϫ/Ϫ cb2 gene (CB1 and CB2 mice) and back-crossed to a A commercially available carbamated amylose ChiralPak-AD C57BL6͞J background were originally obtained from A. Zimmer chiral stationary phase coated on silica support was used (Daicel (Bonn University, Bonn, Germany). Wild-type littermates were Chemical Industries, Tokyo). Column dimensions were 10 ␮m, used as controls. The animal care and the protocols met the 250 ϫ 4.6 mm i.d. The mobile phase was 10% ethanol and 90% guidelines of the Institutional Animal Care and Use Committees. hexane, the flow rate was 1 ml͞min, and the monitoring wavelength for the compound was 205 nm. When a 20-␮l mixture of the two Isolation of ARA-S. Ϸ Fresh bovine brains ( 1.5 kg) were homogenized pairs of D- and L-methylated ARA-S isomers was injected, good in chloroform͞methanol͞water (1:1:0.5), followed by centrifuga- ␣ Ͼ ϫ selectivity (selectivity factor, 1.5) and resolution were obtained tion for 20 min at 7,500 g at 4°C. Nonmiscible residues were (Fig. 1C). filtered, and the organic layer was separated from the water layer and evaporated to dryness. This dry organic extract was redissolved Methylation of ARA-S. Synthetic ARA-S (39.1 mg, 1 mmol) was in methanol, filtered, and evaporated to dryness (extract A). dissolved in dimethylformamide (2 ml), and potassium carbonate (750 mg, 5.42 mmol) was added, some of which remained undis- GC͞MS Analysis. One milligram of extract A was dissolved in 10 ml solved. The mixture was stirred under nitrogen. Then iodomethane of chloroform. Ten microliters of this solution was blown down with ␮ nitrogen, and 10 ␮lofN,O-bis(trimethyl-silyl)trifluoroacetamide, a (100 l, 1.62 mmol) was injected, and the reaction was stirred ␮ overnight. Water (20 ml) was added, and reaction mixture was silylating agent, was added. After 60 min, 1 l of the silylated ϫ mixture was injected into a TRACE GC͞MS 2000 system with extracted with chloroform (3 25 ml). The chloroform extract was ϫ electron impact ionization detector. The column used was ZB-5 washed with brine solution (2 25 ml), dried over MgSO4, filtered, (5% phenyl͞95% dimethylpolysiloxane, 30 ϫ 0.25 mm, 0.25 ␮m). and evaporated. The residue was purified by column chromatog- ͞ The experimental conditions were as follows: inlet, 250°C; detector, raphy by using petroleum ether ether (6:4) as eluent (yield: 77%). 280°C; splitless injection͞purge time, 1 min; initial temperature, NMR and GC͞MS analyses indicated that methylation had taken 70°C; initial time, 5 min; rate, 20°C͞min; final temperature, 280°C. place at the carboxyl position only. Extract A was methylated by the same method. High-Resolution MS Analysis. High-resolution electron impact mass spectra were obtained with an AutoSpec Premier mass spectrom- Tissue Preparation. Isolated mesenteric artery segments were pre- eter (Waters Micromass). After silylation of synthetic ARA-S and pared from male Sprague–Dawley rats (185–200 g), and the con- extract A, as described above, 1 ␮l of the respective solution was tractile responses of phenylephrine precontracted preparations injected into the HRGC-MS instrument under identical experi- were monitored by using a wire myograph under conditions de- mental conditions as described for the low-resolution GC͞MS scribed in ref. 18. Vasodilator responses were expressed as per- analysis (see above). centage relaxation of the phenylephrine-induced tone. The func- tional integrity of the endothelium was verified in all preparations HPLC Analysis. A computer-controlled Waters chromatograph with by Ͼ90% relaxation elicited by 10 ␮M . Endothelial the following components was used: MILLENNIUM software (version denudation was achieved by rubbing the inside of the vessel with a 3.20), 600E multisolvent delivery pump, 20-, 100-, and 200-␮l loops, mounting wire and verified by the loss of the relaxing response to and a Photodiode Array (996 model) detector. All solvents used 10 ␮M acetylcholine (Ͼ10% residual relaxation). Concentration– were HPLC-grade; acetonitrile, hexane, and ethanol were from J.T. response curves were generated by cumulative addition of an ␮ Baker. Doubly distilled water was filtered on a 0.2- m cellulose agonist. Antagonists were added 20 min before the agonist and mixed ester membrane (Schleicher & Schuell). Before injection into remained in the medium throughout the test period. the HPLC, the crude samples were filtered by loading concentrated Segments of the abdominal aorta were from male Sabra rats (300 ␮ portion extracts onto a 0.2- m Gelman nylon syringe filter. g) obtained from the animal facility of Hebrew University. Rats Isolation of ARA-S from extract A was performed on semi- Ϫ1 ␮ ϫ were anesthetized by pentobarbital (50 mg kg i.p.), and the preparative Waters RP-Symmetry Prep C18 column (7- m, 7.8 abdominal aorta was excised and transferred to a dish filled with 300 mm). A 200-␮l aliquot of the crude material dissolved in Krebs–Henseleit solution, cleared of periadventitial tissue, and cut methanol was injected into the column and analyzed by a reversed- into 3-mm-long ring segments. The rings were suspended in a 10-ml phase HPLC. The mobile phase consisted of water (pH 2.6 by organ bath gassed with 5% CO in O and maintained at 37°C by phosphoric acid) and acetonitrile. The gradient conditions were as 2 2 follows: 75% acetonitrile for 15 min, followed by increasing aceto- using two stainless steel hooks, one of which was attached to an nitrile to 100% acetonitrile within 2 min. After 13 min, the isometric force transducer (BIOPAC Systems, Goleta, CA). The concentration of acetonitrile was returned to the starting conditions rings were stretched to a basal tension of 1.0 g and were equilibrated (75%) within 2 min. An additional 15 min were needed to rees- for 60 min with the medium changed every 15–20 min. The segment ␮ tablish the initial equilibrium state. Flow rate was 3 ml͞min, and the was then precontracted by 5 M phenylephrine. Concentration– monitoring wavelength was 205 nm. The ARA-S sharp peak eluted response curves were generated by cumulative addition of the ͞ at almost 13 min. Approximately 25 successive semipreparative agonist. Indomethacin dissolved in 5% wt vol NaHCO3 solution injections isolated Ϸ0.2 mg of ARA-S. Directly after the collection, was added to the organ bath to a final concentration of 10 ␮M. the ARA-S compound was reinjected to an analytical column of Waters Symmetry C18 (5 ␮m, 4.6 ϫ 250 mm). A 100-␮l aliquot was Cell Culture. HUVEC were maintained in primary culture as injected and analyzed by isocratic reversed-phase mode at 30°C. described in ref. 48. Preconfluent cultures (two to five passages) The mobile phase was acetonitrile͞water (pH 2.6 by H3PO4), 75:25 were incubated with vehicle or agonist in the presence or absence (vol͞vol). The flow rate was 1 ml͞min, and the monitoring wave- of an antagonist, as described below. As for the RAW 264.7 length was 205 nm. Synthetic ARA-S eluted at the same retention macrophages, the cells were cultured at 37°C in a humid atmo- time (Ϸ11 min) as the natural compound. The UV spectrum of the sphere of 5% CO2 for6hinDMEM with 10% FCS and all two compounds showed an excellent match. Waters PDA software supplements.

2432 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510676103 Milman et al. Downloaded by guest on October 1, 2021 Ϫ/Ϫ Western Blotting. Phosphorylated and unphosphorylated forms of Plasma TNF-␣ Levels in Endotoxemic Mice. Male C57Bl6͞J, CB1 , Ϫ/Ϫ protein kinase B͞Akt and p42͞44 MAP kinase were visualized and CB2 mice were pretreated with the indicated drug 45 min by Western blotting using appropriate antibodies as described before an i.p. injection of 1 mg͞kg LPS. After an additional 90 min, earlier (48). the mice were decapitated, and trunk blood was collected for determination of TNF-␣ levels. ARA-S and Abn-CBD were dis- Ligand Binding. ARA-S binding to CB1 receptors was assessed in solved in one part DMSO, one part Tween 80, and four parts PBS, displacement assays by using 0.5 nM [3H]CP-55,940 as the and controls were treated with the same vehicle. labeled ligand and 1 ␮M SR141716 to determine nonspecific binding, using crude plasma membranes from mouse cerebellum Reactive Oxygen Intermediate Assay. RAW 264.7 cells, suspended in HBSS without phenol red, were distributed in plastic luminometer (49). ARA-S binding to CB2 receptors was assayed similarly by using 1.0 nM [3H]CP-55,940 and 0.5 ␮M SR144528 in HEK293 tubes. ARA-S was added to the samples followed by addition of 10 ␮l of luminol and 30 ␮l of zymosan (53). The chemiluminescense cells transfected with rat CB2 receptors (50). TRPV1 receptor binding competition assays were done by using CHO cells peak was then recorded by luminometer (Biomate LB 9500 T, transfected with the rat TRPV1 receptor, [3H]resiniferotoxin as Berthold, Wildbad, Germany). the labeled ligand, and 1 ␮M unlabeled resiniferotoxin to NO Determination. NO was determined by measuring the accumu- determine nonspecific binding (51). lated nitrite in the supernatants of ARA-S-treated peritoneal macrophages (52). Murine Macrophages. Peritoneal exudate macrophages from 8- to 9-week-old C57BL͞6 female mice were harvested 4 days after TNF-␣ Determination. TNF-␣ (pg͞ml) in cell culture supernatants or injection of 1.5 ml of 3% thioglycolate medium. Peritoneal mac- in mouse blood plasma was determined by ‘‘sandwich’’ ELISA rophages were cultured in 96-microwell flat bottomed plates. Two technique. ELISA reagents were used according to the manufac- hours later the cells were rinsed to remove unattached cells. After turer’s protocol (R & D Systems). activation with LPS (1 ␮g͞ml), ARA-S diluted to various concen- trations with DMEM was added to the cells. After 24-h incubation, We thank the National Institute on Drug Abuse for Grant DA 9789 (to supernatants were collected and stored at Ϫ20°C until assayed for R.M.). Work done in the U.S. laboratory was supported by intramural TNF-␣ and NO. National Institutes of Health funds.

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