Cannabinoid mechanisms contribute to the therapeutic efficacy of the kratom alkaloid mitragynine against neuropathic, but not inflammatory pain.

AIMS: Mitragynine (MG) is an alkaloid found in Mitragyna speciosa (kratom), a plant used to self-treat symptoms of opioid withdrawal and pain. Kratom products are commonly used in combination with cannabis, with the self-treatment of pain being a primary motivator of use. Both cannabinoids and kratom alkaloids have been characterized to alleviate symptoms in preclinical models of neuropathic pain such as chemotherapy-induced peripheral neuropathy (CIPN). However, the potential involvement of cannabinoid mechanisms in MG's efficacy in a rodent model of CIPN have yet to be explored. MAIN METHODS: Prevention of oxaliplatin-induced mechanical hypersensitivity and formalin-induced nociception were assessed following intraperitoneal administration of MG and CB1, CB2, or TRPV1 antagonists in wildtype and cannabinoid receptor knockout mice. The effects of oxaliplatin and MG exposure on the spinal cord endocannabinoid lipidome was assessed by HPLC-MS/MS. KEY FINDINGS: The efficacy of MG on oxaliplatin-induced mechanical hypersensitivity was partially attenuated upon genetic deletion of cannabinoid receptors, and completely blocked upon pharmacological inhibition of CB1, CB2, and TRPV1 channels. This cannabinoid involvement was found to be selective to a model of neuropathic pain, with minimal effects on MG-induced antinociception in a model of formalin-induced pain. Oxaliplatin was found to selectively disrupt the endocannabinoid lipidome in the spinal cord, which was prevented by repeated MG exposure. SIGNIFICANCE: Our findings suggest that cannabinoid mechanisms contribute to the therapeutic efficacy of the kratom alkaloid MG in a model of CIPN, which may result in increased therapeutic efficacy when co-administered with cannabinoids.

Bioactive Lipids in Cancer, Inflammation and Related Diseases : Acute and Chronic Mild Traumatic Brain Injury Differentially Changes Levels of Bioactive Lipids in the CNS Associated with Headache.

Headache is a common complaint after mild traumatic brain injury (mTBI). Changes in the CNS lipidome were previously associated with acrolein-induced headache in rodents. mTBI caused similar headache-like symptoms in rats; therefore, we tested the hypothesis that mTBI might likewise alter the lipidome. Using a stereotaxic impactor, rats were given either a single mTBI or a series of 4 mTBIs 48 h apart. 72 h later for single mTBI and 7 days later for repeated mTBI, the trigeminal ganglia (TG), trigeminal nucleus (TNC), and cerebellum (CER) were isolated. Using HPLC/MS/MS, ~80 lipids were measured in each tissue and compared to sham controls. mTBI drove widespread alterations in lipid levels. Single mTBI increased arachidonic acid and repeated mTBI increased prostaglandins in all 3 tissue types. mTBI affected multiple TRPV agonists, including N-arachidonoyl ethanolamine (AEA), which increased in the TNC and CER after single mTBI. After repeated mTBI, AEA increased in the TG, but decreased in the TNC. Common to all tissue types in single and repeated mTBI was an increase the AEA metabolite, N-arachidonoyl glycine, a potent activator of microglial migration. Changes in the CNS lipidome associated with mTBI likely play a role in headache and in long-term neurodegenerative effects of repeated mTBI.

Chemotherapy-induced cachexia dysregulates hypothalamic and systemic lipoamines and is attenuated by cannabigerol.

BACKGROUND: Muscle wasting, anorexia, and metabolic dysregulation are common side-effects of cytotoxic chemotherapy, having a dose-limiting effect on treatment efficacy, and compromising quality of life and mortality. Extracts of Cannabis sativa, and analogues of the major phytocannabinoid Δ9-tetrahydrocannabinol, have been used to ameliorate chemotherapy-induced appetite loss and nausea for decades. However, psychoactive side-effects limit their clinical utility, and they have little efficacy against weight loss. We recently established that the non-psychoactive phytocannabinoid cannabigerol (CBG) stimulates appetite in healthy rats, without neuromotor side-effects. The present study assessed whether CBG attenuates anorexia and/or other cachectic effects induced by the broad-spectrum chemotherapy agent cisplatin. METHODS: An acute cachectic phenotype was induced in adult male Lister-hooded rats by 6 mg/kg (i.p.) cisplatin. In total 66 rats were randomly allocated to groups receiving vehicle only, cisplatin only, or cisplatin and 60 or 120 mg/kg CBG (po, b.i.d.). Feeding behavior, bodyweight and locomotor activity were recorded for 72 hours, at which point rats were sacrificed for post-mortem analyses. Myofibre atrophy, protein synthesis and autophagy dysregulation were assessed in skeletal muscle, plasma metabolic profiles were obtained by untargeted 1H-NMR metabonomics, and levels of endocannabinoid-like lipoamines quantified in plasma and hypothalami by targeted HPLC-MS/MS lipidomics. RESULTS: CBG (120 mg/kg) modestly increased food intake, predominantly at 36-60hrs (p<0.05), and robustly attenuated cisplatin-induced weight loss from 6.3% to 2.6% at 72hrs (p<0.01). Cisplatin-induced skeletal muscle atrophy was associated with elevated plasma corticosterone (3.7 vs 13.1ng/ml, p<0.01), observed selectively in MHC type IIx (p<0.05) and IIb (p<0.0005) fibres, and was reversed by pharmacological rescue of dysregulated Akt/S6-mediated protein synthesis and autophagy processes. Plasma metabonomic analysis revealed cisplatin administration produced a wide-ranging aberrant metabolic phenotype (Q2Y=0.5380, p=0.001), involving alterations to glucose, amino acid, choline and lipid metabolism, citrate cycle, gut microbiome function, and nephrotoxicity, which were partially normalized by CBG treatment (Q2Y=0.2345, p=0.01). Lipidomic analysis of hypothalami and plasma revealed extensive cisplatin-induced dysregulation of central and peripheral lipoamines (29/79 and 11/26 screened, respectively), including reversible elevations in systemic N-acyl glycine concentrations which were negatively associated with the anti-cachectic effects of CBG treatment. CONCLUSIONS: Endocannabinoid-like lipoamines may have hitherto unrecognized roles in the metabolic side-effects associated with chemotherapy, with the N-acyl glycine subfamily in particular identified as a potential therapeutic target and/or biomarker of anabolic interventions. CBG-based treatments may represent a novel therapeutic option for chemotherapy-induced cachexia, warranting investigation in tumour-bearing cachexia models.

Cannabidiol's Upregulation of N-acyl Ethanolamines in the Central Nervous System Requires N-acyl Phosphatidyl Ethanolamine-Specific Phospholipase D.

Introduction: Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are bioactive cannabinoids. We recently showed that acute THC administration drives region-dependent changes in the mouse brain lipidome. This study tested the hypothesis that cell lines representing cell types present in the central nervous system (CNS), neurons (N18 cells), astrocytes (C6 glioma cells), and microglia (BV2 cells) would respond differently to THC, CBD, or their combination. This experimental strategy also allowed us to test the hypothesis that THC and CBD are metabolized differently if presented in combination and to test the hypothesis that responses to CBD are not like the fatty acid amide hydrolase (FAAH) inhibitor URB597. Finally, we tested the hypothesis that CBD's CNS effects would differ in the N-acyl phosphatidyl ethanolamine-specific phospholipase D (NAPE-PLD) knockout (KO) compared to wild-type (WT) mice. Methods: N18, C6, and BV2 cells were stimulated with 1 µM THC, 1 µM CBD, 1 µM THC:CBD, 1 µM URB597, or vehicle for 2 h and lipids extracted. Adult female WT and NAPE-PLD KO mice were injected with 3 mg/kg CBD or vehicle i.p., brains collected 2 h later, eight brain regions dissected, and lipids extracted. Extracted lipids were characterized and quantified using high-pressure liquid chromatography coupled with tandem mass spectrometry (HPLC/MS/MS). Results: Lipid levels in each cell type were differentially affected by THC, CBD, or THC:CBD with a few exceptions. In all cell lines, THC increased levels of arachidonic acid and CBD increased levels of N-acyl ethanolamines (NAEs), including N-arachidonoyl ethanolamine. More THC remained when cells were coincubated with CBD; however, levels of THC metabolites were cell-type dependent. CBD and URB597 caused very different lipid profiles in the cell-based assays with the primary similarity being increases in NAEs. CBD increased levels of NAEs in the WT hippocampus, cerebellum, thalamus, cortex, midbrain, and brainstem; however, NAEs did not increase in any brain region after CBD in NAPE-PLD KO mice. Conclusions: CBD and THC differentially modify the lipidome of the brain and CNS-type cell lines. Increases in NAEs observed after CBD treatment had previously been attributed to FAAH inhibition; however, data here suggest the alternative hypothesis that CBD is activating NAPE-PLD to increase NAE levels.

Prostaglandin levels, vaginal innervation, and cyst innervation as peripheral contributors to endometriosis-associated vaginal hyperalgesia in rodents.

Endometriosis is a painful condition characterized by growth of endometrial cysts outside the uterus. Here, we tested the hypothesis that peripheral innervation and prostaglandin levels contribute to endometriosis-associated pain. Female Sprague-Dawley rats (n = 16) were surgically instrumented by transplanting uterine tissue onto mesenteric arteries within the peritoneal cavity to create a model of endometriosis which forms extra-uterine endometrial cysts and vaginal hyperalgesia. Our results describe a significant positive correlation between endometriosis-induced vaginal hyperalgesia and cyst innervation density (sensory, r = 0.70, p = 0.003; sympathetic, r = 0.55, p = 0.03), vaginal canal sympathetic innervation density (r = 0.80, p = 0.003), and peritoneal fluid levels of the prostaglandins PGE2 (r = 0.65, p = 0.01) and PGF2α (r = 0.63, p = 0.02). These results support the involvement of cyst innervation and prostaglandins in endometriosis-associated pain. We also describe how sympathetic innervation density of the vaginal canal is an important predictor of vaginal hyperalgesia.

p53 coordinates decidual sestrin 2/AMPK/mTORC1 signaling to govern parturition timing.

Inflammation and oxidative stress are known risk factors for preterm birth (PTB); however, the mechanisms and pathways that influence this condition are not fully described. Previously, we showed that mTORC1 signaling is increased in mice harboring a uterine-specific deletion of transformation-related protein 53 (p53d/d mice), which exhibit premature decidual senescence that triggers spontaneous and inflammation-induced PTB. Treatment with the mTORC1 inhibitor rapamycin reduced the incidence of PTB in the p53d/d mice. Decidual senescence with heightened mTORC1 signaling is also a signature of human PTB. Here, we have identified an underlying mechanism for PTB and a potential therapeutic strategy for treating the condition. Treatment of pregnant p53d/d mice with either the antidiabetic drug metformin or the antioxidant resveratrol activated AMPK signaling and inhibited mTORC1 signaling in decidual cells. Both metformin and resveratrol protected against spontaneous and inflammation-induced PTB in p53d/d females. Using multiple approaches, we determined that p53 interacts with sestrins to coordinate an inverse relationship between AMPK and mTORC1 signaling that determines parturition timing. This signature was also observed in human decidual cells. Together, these results reveal that p53-dependent coordination of AMPK and mTORC1 signaling controls parturition timing and suggest that metformin and resveratrol have therapeutic potential to prevent PTB.

Levels of bioactive lipids in cooking oils: olive oil is the richest source of oleoyl serine.

BACKGROUND: Rates of osteoporosis are significantly lower in regions of the world where olive oil consumption is a dietary cornerstone. Olive oil may represent a source of oleoyl serine (OS), which showed efficacy in animal models of osteoporosis. Here, we tested the hypothesis that OS as well as structurally analogous N-acyl amide and 2-acyl glycerol lipids are present in the following cooking oils: olive, walnut, canola, high heat canola, peanut, safflower, sesame, toasted sesame, grape seed, and smart balance omega. METHODS: Methanolic lipid extracts from each of the cooking oils were partially purified on C-18 solid-phase extraction columns. Extracts were analyzed with high-performance liquid chromatography-tandem mass spectrometry, and 33 lipids were measured in each sample, including OS and bioactive analogs. RESULTS: Of the oils screened here, walnut oil had the highest number of lipids detected (22/33). Olive oil had the second highest number of lipids detected (20/33), whereas grape-seed and high-heat canola oil were tied for lowest number of detected lipids (6/33). OS was detected in 8 of the 10 oils tested and the levels were highest in olive oil, suggesting that there is something about the olive plant that enriches this lipid. CONCLUSIONS: Cooking oils contain varying levels of bioactive lipids from the N-acyl amide and 2-acyl glycerol families. Olive oil is a dietary source of OS, which may contribute to lowered prevalence of osteoporosis in countries with high consumption of this oil.

Lipopolysaccharide-induced murine embryonic resorption involves changes in endocannabinoid profiling and alters progesterone secretion and inflammatory response by a CB1-mediated fashion.

Genital tract infections are a common complication of human pregnancy that can result in miscarriage. We have previously shown that a lipopolysaccharide (LPS) induces embryonic resorption in a murine model of inflammatory miscarriage. This is accompanied by a dramatic decrease in systemic progesterone levels associated with a robust pro-inflammatory response that results in embryo resorption. Here, we tested the hypothesis that the endogenous cannabinoid system (eCS), through cannabinoid receptor 1 (CB1), plays a role in regulating progesterone levels and, therefore, the pro-inflammatory response. We show that LPS treatment in pregnant mice causes significant changes in the eCS ligands, which are reversed by progesterone treatment. We further show the CB1-KO mice maintain higher plasma progesterone levels after LPS treatment, which is associated with a feebler uterine inflammatory response and a significant drop in embryo resorption. These data suggest that manipulation of CB1 receptors and/or ligands is a potential therapeutic avenue to decrease infection-induced miscarriage.

Alterations in endocannabinoid tone following chemotherapy-induced peripheral neuropathy: effects of endocannabinoid deactivation inhibitors targeting fatty-acid amide hydrolase and monoacylglycerol lipase in comparison to reference analgesics following cisplatin treatment.

Cisplatin, a platinum-derived chemotherapeutic agent, produces mechanical and coldallodynia reminiscent of chemotherapy-induced neuropathy in humans. The endocannabinoid system represents a novel target for analgesic drug development. The endocannabinoid signaling system consists of endocannabinoids (e.g. anandamide (AEA) and 2-arachidonoylglycerol (2-AG)), cannabinoid receptors (e.g. CB(1) and CB(2)) and the enzymes controlling endocannabinoid synthesis and degradation. AEA is hydrolyzed by fatty-acid amide hydrolase (FAAH) whereas 2-AG is hydrolyzed primarily by monoacylglycerol lipase (MGL). We compared effects of brain permeant (URB597) and impermeant (URB937) inhibitors of FAAH with an irreversible inhibitor of MGL (JZL184) on cisplatin-evoked behavioral hypersensitivities. Endocannabinoid modulators were compared with agents used clinically to treat neuropathy (i.e. the opioid analgesic morphine, the anticonvulsant gabapentin and the tricyclic antidepressant amitriptyline). Cisplatin produced robust mechanical and cold allodynia but did not alter responsiveness to heat. After neuropathy was fully established, groups received acute intraperitoneal (i.p.) injections of vehicle, amitriptyline (30 mg/kg), gabapentin (100 mg/kg), morphine (6 mg/kg), URB597 (0.1 or 1 mg/kg), URB937 (0.1 or 1 mg/kg) or JZL184 (1, 3 or 8 mg/kg). Pharmacological specificity was assessed by coadministering each endocannabinoid modulator with either a CB(1) (AM251 3 mg/kg), CB(2) (AM630 3 mg/kg), TRPV1 (AMG9810 3 mg/kg) or TRPA1 (HC030031 8 mg/kg) antagonist. Effects of cisplatin on endocannabinoid levels and transcription of receptors (CB(1), CB(2), TRPV1, TRPA1) and enzymes (FAAH, MGL) linked to the endocannabinoid system were also assessed. URB597, URB937, JZL184 and morphine reversed cisplatin-evoked mechanical and cold allodynia to pre-cisplatin levels. By contrast, gabapentin only partially reversed the observed allodynia while amitriptyline, administered acutely, was ineffective. CB(1) or CB(2) antagonists completely blocked the anti-allodynic effects of both FAAH (URB597, URB937) and MGL (JZL184) inhibitors to mechanical and cold stimulation. By contrast, the TRPV1 antagonist AMG9810 blocked the anti-allodynic efficacy of both FAAH inhibitors, but not the MGL inhibitor. By contrast, the TRPA1 antagonist HC30031 did not attenuate anti-allodynic efficacy of any endocannabinoid modulator. When the levels of endocannabinoids were examined, cisplatin increased both anandamide (AEA) and 2-arachidonoylglycerol (2-AG) levels in the lumbar spinal cord and decreased 2-AG levels (but not AEA) in dorsal hind paw skin. RT-PCR showed that mRNA for FAAH, but not other markers, was upregulated by cisplatin treatment in lumbar spinal cord. The present studies demonstrate that cisplatin alters endocannabinoid tone and that inhibition of endocannabinoid hydrolysis alleviates chemotherapy-induced mechanical and cold allodynia. The anti-allodynic effects of FAAH and MGL inhibitors are mediated by CB(1) and CB(2) cannabinoid receptors, whereas TRPV1, but not TRPA1, -dependent mechanisms contribute to the anti-allodynic efficacy of FAAH (but not MGL) inhibitors. Strikingly, endocannabinoid modulators potently suppressed cisplatin-evoked allodynia with a rapid onset and showed efficacy that equaled or exceeded that of major classes of anti-neuropathic pain medications used clinically. Thus, inhibition of endocannabinoid hydrolysis, via FAAH or MGL inhibitors, represents an efficacious pharmacological approach for suppressing chemotherapy-induced neuropathic pain.

Δ(9) -Tetrahydrocannabinol and N-arachidonyl glycine are full agonists at GPR18 receptors and induce migration in human endometrial HEC-1B cells.

BACKGROUND AND PURPOSE: Endometriosis is a disorder in which the endometrium forms growths outside the uterus and is associated with chronic pain. Recent evidence suggests that endometrial motility plays a role in the aetiology of endometriosis. The endocannabinoid system regulates cellular migration. Given the growing involvement of the endocannabinoids in reproduction, we investigated the role of the endocannabinoid system in migration of endometrial cells. EXPERIMENTAL APPROACH: Migration of the human endometrial HEC-1B cells was assayed. Standard PCR techniques were used to determine the presence of the GPCR, GPR18, in HEC-1B cells, and p44/42 MAPK was assayed in stably transfected HEK293-GPR18 cells to determine receptor specificity for known cannabinoid agonists and antagonists. N-arachidonoyl ethanolamine (AEA) metabolism was measured, using HPLC/MS/MS for lipid analysis. KEY RESULTS: AEA, Δ(9) -tetrahydrocannabinol (Δ(9) -THC) and N-arachidonoyl glycine (NAGly) induce migration of HEC-1B cells through cannabinoid CB(1) receptor-independent mechanisms. MAPK activation in HEK293-GPR18 cells revealed novel pharmacology for known CB(1) and CB(2) receptor ligands at GPR18 receptors, including Δ(9) -THC, which activates MAPK at nanomolar concentrations, whereas WIN 55212-2, CP55940, JWH-133 and JWH-015, and arachidonyl-1-hydroxy-2-propylamide (R1-methanandamide) had no effect. Moreover, HEC-1B migration and MAPK activation by NAGly and Δ(9) -THC were antagonized by Pertussis toxin, AM251 and cannabidiol. CONCLUSIONS AND IMPLICATIONS: An understanding of the function and regulation of GPR18 and its molecular interactions with endogenous ligands, and how phytocannabinoids play a role with GPR18 signalling is vital if we are to comprehensively assess the function of the cannabinoid signalling system in human health and disease. LINKED ARTICLES: This article is commented on by Alexander, pp. 2411-2413 of this issue and is part of a themed section on Cannabinoids in Biology and Medicine. To view Alexander visit http://dx.doi.org/10.1111/j.1476-5381.2011.01731.x. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Oleoyl serine, an endogenous N-acyl amide, modulates bone remodeling and mass.

Bone mass is determined by a continuous remodeling process, whereby the mineralized matrix is being removed by osteoclasts and subsequently replaced with newly formed bone tissue produced by osteoblasts. Here we report the presence of endogenous amides of long-chain fatty acids with amino acids or with ethanolamine (N-acyl amides) in mouse bone. Of these compounds, N-oleoyl-l-serine (OS) had the highest activity in an osteoblast proliferation assay. In these cells, OS triggers a Gi-protein-coupled receptor and Erk1/2. It also mitigates osteoclast number by promoting osteoclast apoptosis through the inhibition of Erk1/2 phosphorylation and receptor activator of nuclear-κB ligand (RANKL) expression in bone marrow stromal cells and osteoblasts. In intact mice, OS moderately increases bone volume density mainly by inhibiting bone resorption. However, in a mouse ovariectomy (OVX) model for osteoporosis, OS effectively rescues bone loss by increasing bone formation and markedly restraining bone resorption. The differential effect of exogenous OS in the OVX vs. intact animals is apparently a result of an OVX-induced decrease in skeletal OS levels. These data show that OS is a previously unexplored lipid regulator of bone remodeling. It represents a lead to antiosteoporotic drug discovery, advantageous to currently available therapies, which are essentially either proformative or antiresorptive.

Uterine-specific p53 deficiency confers premature uterine senescence and promotes preterm birth in mice.

Many signaling pathways that contribute to tumorigenesis are also functional in pregnancy, although they are dysregulated in the former and tightly regulated in the latter. Transformation-related protein 53 (Trp53), which encodes p53, is a tumor suppressor gene whose mutation is strongly associated with cancer. However, its role in normal physiological processes, including female reproduction, is poorly understood. Mice that have a constitutive deletion of Trp53 exhibit widespread development of carcinogenesis at early reproductive ages, compromised spermatogenesis, and fetal exencephaly, rendering them less amenable to studying the role of p53 in reproduction. To overcome this obstacle, we generated mice that harbor a conditional deletion of uterine Trp53 and examined pregnancy outcome in females with this genotype. These mice had normal ovulation, fertilization, and implantation; however, postimplantation uterine decidual cells showed terminal differentiation and senescence-associated growth restriction with increased levels of phosphorylated Akt and p21, factors that are both known to participate in these processes in other systems. Strikingly, uterine deletion of Trp53 increased the incidence of preterm birth, a condition that was corrected by oral administration of the selective COX2 inhibitor celecoxib. We further generated evidence to suggest that deletion of uterine Trp53 induces preterm birth through a COX2/PGF synthase/PGF(2alpha) pathway. Taken together, our observations underscore what we believe to be a new critical role of uterine p53 in parturition.

The endocannabinoid anandamide is a precursor for the signaling lipid N-arachidonoyl glycine by two distinct pathways.

BACKGROUND: N-arachidonoyl glycine (NAGly) is an endogenous signaling lipid with a wide variety of biological activity whose biosynthesis is poorly understood. Two primary biosynthetic pathways have been proposed. One suggests that NAGly is formed via an enzymatically regulated conjugation of arachidonic acid (AA) and glycine. The other suggests that NAGly is an oxidative metabolite of the endogenous cannabinoid, anandamide (AEA), through an alcohol dehydrogenase. Here using both in vitro and in vivo assays measuring metabolites with LC/MS/MS we test the hypothesis that both pathways are present in mammalian cells. RESULTS: The metabolic products of deuterium-labeled AEA, D4AEA (deuterium on ethanolamine), indicated that NAGly is formed by the oxidation of the ethanolamine creating a D2NAGly product in both RAW 264.7 and C6 glioma cells. Significantly, D4AEA produced a D0NAGly product only in C6 glioma cells suggesting that the hydrolysis of AEA yielded AA that was used preferentially in a conjugation reaction. Addition of the fatty acid amide (FAAH) inhibitor URB 597 blocked the production of D0NAGly in these cells. Incubation with D8AA in C6 glioma cells likewise produced D8NAGly; however, with significantly less efficacy leading to the hypothesis that FAAH-initiated AEA-released AA conjugation with glycine predominates in these cells. Furthermore, the levels of AEA in the brain were significantly increased, whereas those of NAGly were significantly decreased after systemic injection of URB 597 in rats and in FAAH KO mice further supporting a role for FAAH in endogenous NAGly biosynthesis. Incubations of NAGly and recombinant FAAH demonstrated that NAGly is a significantly less efficacious substrate for FAAH with only ~50% hydrolysis at 30 minutes compared to 100% hydrolysis of AEA. Co-incubations of AEA and glycine with recombinant FAAH did not, however, produce NAGly. CONCLUSION: These data support the hypothesis that the signaling lipid NAGly is a metabolic product of AEA by both oxidative metabolism of the AEA ethanolamine moiety and through the conjugation of glycine to AA that is released during AEA hydrolysis by FAAH.

Potential anti-inflammatory actions of the elmiric (lipoamino) acids.

A library of amino acid-fatty acid conjugates (elmiric acids) was synthesized and evaluated for activity as potential anti-inflammatory agents. The compounds were tested in vitro for their effects on cell proliferation and prostaglandin production, and compared with their effects on in vivo models of inflammation. LPS stimulated RAW 267.4 mouse macrophage cells were the in vitro model and phorbol ester-induced mouse ear edema served as the principal in vivo model. The prostaglandin responses were found to be strongly dependent on the nature of the fatty acid part of the molecule. Polyunsaturated acid conjugates produced a marked increase in media levels of i15-deoxy-PGJ(2) with minimal effects on PGE production. It is reported in the literature that prostaglandin ratios in which the J series predominates over the E series promote the resolution of inflammatory conditions. Several of the elmiric acids tested here produced such favorable ratios suggesting that their potential anti-inflammatory activity occurs via a novel mechanism of action. The ear edema assay results were generally in agreement with the prostaglandin assay findings indicating a connection between them.

The influence of ovariectomy with or without estrogen replacement on responses of rat gracile nucleus neurons to stimulation of hindquarter skin and pelvic viscera.

Responses of neurons in the gracile nucleus (NG) of female rats to tactile and visceral stimulation change across the estrous cycle [J. Neurosci. 20 (2000) 7722]. To investigate estrogen's role in these changes, responses of NG neurons to tactile and visceral stimuli were examined in three groups: ovariectomized (OVX), OVX with estrogen replacement (OVX+E2), or sham OVX (tested in diestrus; shamOVX-D). The stimuli were: gentle brushing of hindquarter skin, pressure on the cervix, and distention of the uterus, vagina, or colon. After OVX, the magnitude of multi-unit responses to brushing the perineum, hip and tail, but not the foot and leg, were significantly reduced relative to shamOVX-D. OVX+E2 restored this magnitude to the same level as shamOVX-D, but not, as expected, to levels as large as previously observed in proestrus. After OVX, responses of single neurons to stimulation of the uterus, cervix, and colon were more likely to be excitatory (versus inhibitory) than they had been in cycling rats in proestrus (uterus, cervix) or diestrus (colon); OVX+E2 did not restore the inhibitory responses. In contrast, whereas all responses to vaginal distention after OVX were also excitatory, OVX+E2 in this case significantly restored the inhibitory responses. These findings provide further support for the conclusion that response characteristics of NG neurons are influenced by the rat's hormonal milieu, but also indicate that the influences are not a simple reflection of estrogen levels. The findings further suggest that NG is a component of neural systems that contribute to both reproductive behaviors and vaginal nociception.

Estrogen replacement reverses ovariectomy-induced vaginal hyperalgesia in the rat.

OBJECTIVES: The loss of ovarian function in women through aging or oophorectomy is often associated with the development of vaginal hyperalgesia that can be alleviated with estrogen replacement. This study examined if ovariectomy in rats would similarly give rise to vaginal hyperalgesia, and, if so, whether estrogen replacement would alleviate it. METHODS: Female rats were trained to perform an operant response to escape vaginal distention delivered by inflating a balloon located in mid-vaginal canal. Percent escape responses to eight different volumes of distention measured in normally cycling rats were compared with measures made in the same rats following ovariectomy (OVX) or sham ovariectomy (shamOVX), and then, in the OVX group, estrogen replacement (OVX+E2). Pressures exerted by the eight volumes on the vaginal wall were also measured, thereby permitting assessment of vaginal tone. RESULTS: Whereas overall escape response percentages after OVX, but not shamOVX, were significantly higher to the largest six distention volumes compared with responses during cycling, there were individual differences in the amount of hyperalgesia. Following OVX+E2, escape response percentages decreased in all but one rat. Vaginal tone after OVX, shamOVX or OVX+E2 did not differ from overall vaginal tone in cycling rats. CONCLUSIONS: Ovariectomy in rats evokes a variable amount of vaginal hyperalgesia that can be alleviated by estrogen replacement in most cases. Thus, the ovariectomized rat appears to provide a useful model for the study of mechanisms underlying the dyspareunia that is associated with loss of ovarian function in women.