Philip J. Kingsley

Aberrant cannabinoid signaling impairs oviductal transport of embryos

Ectopic pregnancy is a major reproductive health issue. Although other underlying causes remain largely unknown, one cause of ectopic pregnancy is embryo retention in the fallopian tube. Here we show that genetic or pharmacologic silencing of cannabinoid receptor CB1 causes retention of a large number of embryos in the mouse oviduct, eventually leading to pregnancy failure. This is reversed by isoproterenol, a β-adrenergic receptor agonist. Impaired oviductal embryo transport is also observed in wild-type mice treated with methanandamide. Collectively, the results suggest that aberrant cannabinoid signaling impedes coordinated oviductal smooth muscle contraction and relaxation crucial to normal oviductal embryo transport. Colocalization of CB1 and β2-adrenergic receptors in the oviduct muscularis implies that a basal endocannabinoid tone in collaboration with adrenergic receptors coordinates oviductal motility for normal journey of embryos into the uterus. Besides uncovering a new regulatory mechanism, this study could be clinically relevant to ectopic pregnancy.

N-Acylphosphatidylethanolamine-hydrolyzing Phospholipase D Is an Important Determinant of Uterine Anandamide Levels during Implantation

Implantation requires reciprocal interaction between blastocysts and a receptive uterus. In mice, one important player in this dialogue involves endocannabinoid signaling via cannabinoid receptor CB1. Anandamide is an endogenous cannabinoid ligand, and its levels are spatiotemporally regulated in the uterus during early pregnancy, showing lower levels in the receptive uterus and at the implantation site. However, the mechanism by which differential uterine anandamide gradients are established under different pregnancy status is not clearly understood. Using multiple approaches, we show here that uterine anandamide levels conducive to implantation are primarily regulated by spatiotemporal expression of Nape-Pld, the gene encoding N-acylphosphatidylethanolamine-hydrolyzing phospholipase D that generates anandamide. The expression is well correlated with its activity and anandamide levels. This study is clinically relevant, since elevated anandamide levels in peripheral circulation are associated with spontaneous pregnancy failure in women.

Selective Visualization of Cyclooxygenase-2 in Inflammation and Cancer by Targeted Fluorescent Imaging Agents

Effective diagnosis of inflammation and cancer by molecular imaging is challenging because of interference from nonselective accumulation of the contrast agents in normal tissues. Here, we report a series of novel fluorescence imaging agents that efficiently target cyclooxygenase-2 (COX-2), which is normally absent from cells, but is found at high levels in inflammatory lesions and in many premalignant and malignant tumors. After either i.p. or i.v. injection, these reagents become highly enriched in inflamed or tumor tissue compared with normal tissue and this accumulation provides sufficient signal for in vivo fluorescence imaging. Further, we show that only the intact parent compound is found in the region of interest. COX-2-specific delivery was unambiguously confirmed using animals bearing targeted deletions of COX-2 and by blocking the COX-2 active site with high-affinity inhibitors in both in vitro and in vivo models. Because of their high specificity, contrast, and detectability, these fluorocoxibs are ideal candidates for detection of inflammatory lesions or early-stage COX-2-expressing human cancers, such as those in the esophagus, oropharynx, and colon.

Repeated homotypic stress elevates 2-arachidonoylglycerol levels and enhances short-term endocannabinoid signaling at inhibitory synapses in basolateral amygdala.

Psychosocial stress is a risk factor for development and exacerbation of neuropsychiatric illness. Repeated stress causes biochemical adaptations in endocannabinoid (eCB) signaling that contribute to stress–response habituation, however, the synaptic correlates of these adaptations have not been examined. Here, we show that the synthetic enzyme for the eCB 2-arachidonoylglycerol (2-AG), diacylglycerol (DAG) lipase alpha, is heterogeneously expressed in the amygdala, and that levels of 2-AG and precursor DAGs are increased in the basolateral amygdala (BLA) after 10 days, but not 1 day, of restraint stress. In contrast, arachidonic acid was decreased after both 1 and 10 days of restraint stress. To examine the synaptic correlates of these alterations in 2-AG metabolism, we used whole-cell electrophysiology to determine the effects of restraint stress on depolarization-induced suppression of inhibition (DSI) in the BLA. A single restraint stress exposure did not alter DSI compared with control mice. However, after 10 days of restraint stress, DSI duration, but not magnitude, was significantly prolonged. Inhibition of 2-AG degradation with MAFP also prolonged DSI duration; the effects of repeated restraint stress and MAFP were mutually occlusive. These data indicate that exposure to repeated, but not acute, stress produces neuroadaptations that confer BLA neurons with an enhanced capacity to elevate 2-AG content and engage in 2-AG-mediated short-term retrograde synaptic signaling. We suggest stress-induced enhancement of eCB-mediated suppression of inhibitory transmission in the BLA could contribute to affective dysregulation associated with chronic stress.

Analysis of endocannabinoids by Ag+ coordination tandem mass spectrometry

The neutral arachidonic acid derivatives N-arachidonylethanolamide (anandamide or AEA) and 2-arachidonylglycerol (2-AG) have been identified as endogenous ligands for the cannabinoid receptors. Quantitation of these endocannabinoids from various tissues has been shown to be essential in the elucidation of cannabinoid-mediated processes in vivo. Here, we describe a novel method for the detection and quantitation of AEA and 2-AG from mammalian tissue. We exploit the ability of silver cation to bind to the polyunsaturated arachidonate backbone of both molecules to form the charged species [M+Ag](+). These complexes are amenable to liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, resulting in the simple and specific quantitation of AEA and 2-AG. The limits of detection of 2-AG and AEA are 13 and 14fmol, respectively, on-column. This method provides an alternative to existing methods, which employ derivation and/or selected ion monitoring (when mass spectrometric detection is used), and may facilitate the understanding of the physiological roles of this new class of compounds.

Selective oxygenation of N-arachidonylglycine by cyclooxygenase-2

Nonsteroidal anti-inflammatory drugs prevent hyperalgesia and inflammation by inhibiting the cyclooxygenase-2 (COX-2) catalyzed oxygenation of arachidonic acid to prostaglandin (PG) H(2). The lipoamino acid N-arachidonylglycine (NAGly) has also been shown to suppress tonic inflammatory pain and is naturally present at significant levels in many of the same mammalian tissues that express COX-2. Here, we report that COX-2 selectively metabolizes NAGly to PGH(2) glycine (PGH(2)-Gly) and hydroxyeicosatetraenoic glycine (HETE-Gly). Site-directed mutagenesis experiments identify the side pocket residues of COX-2, especially Arg-513, as critical determinants of the COX-2 selectivity towards NAGly. This is the first report of a charged arachidonyl derivative that is a selective substrate for COX-2. These results suggest a possible role for COX-2 in the regulation of NAGly levels and the formation of a novel class of eicosanoids from NAGly metabolism.

Fluorinated Cyclooxygenase-2 Inhibitors as Agents in PET Imaging of Inflammation and Cancer

COX-2 is a major contributor to the inflammatory response and cancer progression so it is an important target for prevention and therapy. COX-2 is absent or expressed at low levels in most epithelial cells but is found at high levels in inflammatory lesions, and many premalignant and malignant tumors. Thus, it is an attractive target for molecular imaging. We report a series of novel fluorinated imaging agents, derived from indomethacin or celecoxib that selectively inhibit COX-2. The most promising lead, compound 7, was a fluorinated derivative of celecoxib. Kinetic analysis revealed that this fluorinated compound is a slow, tight-binding inhibitor of COX-2 and exhibits minimal inhibitory activity against COX-1. Efficient incorporation of 18F into compound 7 by radiochemical synthesis and intravenous injection provided sufficient signal for in vivo positron emission tomography (PET) imaging. Selective uptake of 18F-7 was observed in inflamed rat paws compared with the noninflamed contralateral paws and uptake was blocked by pretreatment with the COX-2 inhibitor, celecoxib. Uptake of 18F-7 was not observed when inflammation was induced in COX-2–null mice. In nude mice bearing both a COX-2–expressing human tumor xenograft (1483) and a COX-2–negative xenograft (HCT116), 18F-7 selectively accumulated in the COX-2–expressing tumor. Accumulation was blocked by pretreatment of the animals with celecoxib. The in vitro and in vivo properties of compound 7 suggest it will be a useful probe for early detection of cancer and for evaluation of the COX-2 status of premalignant and malignant tumors. Cancer Prev Res; 4(10); 1536–45. ©2011 AACR.

Genetic Loss of Faah Compromises Male Fertility in Mice

Abstract Marijuana is the most commonly used illicit drug. Although there is some indication that reproductive functions in males are impaired in chronic marijuana users, the genetic evidence and underlying causes remain largely unknown. Herein we show that genetic loss of Faah, which encodes fatty acid amide hydrolase (FAAH), results in elevated levels of anandamide, an endocannabinoid, in the male reproductive system, leading to compromised fertilizing capacity of sperm. This defect is rescued by superimposing deletion of cannabinoid receptor 1 (Cnr1). Retention of Faah−/− sperm on the egg zona pellucida provides evidence that the capacity of sperm to penetrate the zona barrier is hampered by elevated anandamide levels. Collectively, the results show that aberrant endocannabinoid signaling via CNR1 impairs normal sperm function. Besides unveiling a new regulatory mechanism of sperm function, this study has clinical significance in male fertility.

Induction and Function of Lipocalin Prostaglandin D Synthase in Host Immunity

Although mainly expressed in neuronal cells, lipocalin-type PGD synthase (L-PGDS) is detected in the macrophages infiltrated to atherosclerotic plaques. However, the regulation and significance of L-PGDS expression in macrophages are unknown. Here, we found that treatment of macrophages with bacterial endotoxin (LPS) or Pseudomonas induced L-PGDS expression. Epigenetic suppression of L-PGDS expression in macrophages blunted a majority of PGD2 produced after LPS treatment. Chromatin immunoprecipitation assays show that L-PGDS induction was regulated positively by AP-1, but negatively by p53. L-PGDS expression was detected in whole lung and alveolar macrophages treated with LPS or Pseudomonas. L-PGDS overexpressing transgenic mice improved clearance of Pseudomonas from the lung compared with nontransgenic mice. Similarly, intratracheal instillation of PGD2 enhanced removal of Pseudomonas from the lung in mice. In contrast, L-PGDS knockout mice were impaired in their ability to remove Pseudomonas from the lung. Together, our results identify induction of L-PGDS expression by inflammatory stimuli or bacterial infection, the regulatory mechanism of L-PGDS induction, and the protective role of L-PGDS expression in host immune response. Our study suggests a potential therapeutic usage of L-PGDS or PGD2 against Pseudomonas pneumonia.

NMDA-induced Seizure Intensity is Enhanced in COX-2 Deficient Mice

Pharmacological inhibition or genetic deletion of cyclooxygenase (COX)-2, but not COX-1, has been shown to increase susceptibility to kainic acid (KA)-induced excitotoxicity. However, it is unclear if susceptibility to excitotoxins that act through other neurotransmitter receptors is altered by COX-2 inhibition. To further understand the involvement of COX-2 in regulating susceptibility to excitotoxicity, we investigated the effect of COX-2 deletion on excitotoxicity induced by peripheral injection of N-methyl-d-aspartate (NMDA, a specific agonist of the NMDA receptors) or lindane (a GABA(A) receptor antagonist). COX-2(-/-) mice injected intraperitoneally with NMDA (50-100mg/kg) exhibited significantly increased median seizure intensity when compared to COX-2(+/+) mice. Further, COX-2(-/-) mice exposed to NMDA showed neuronal damage, detected by Fluoro Jade B (FJB) staining, in the CA3 region of the hippocampus. There was no FJB staining nor any significant difference in median or maximal seizure intensity in COX-2(+/+) and COX-2(-/-) mice exposed to lindane. LC-MS/MS analysis of brain prostaglandin profile in COX-2(-/-) mice demonstrated a significant increase in PGF(2alpha), TXB(2), PGE(2) and PGD(2) expression 1h after administration of an excitotoxic dose of KA, but not of NMDA. Our findings demonstrate that COX-2 regulates susceptibility to KA and NMDA excitotoxicity, which directly activate glutamatergic neurotransmission, but not to lindane, which indirectly alters glutamatergic neurotransmission. Furthermore, increased levels of prostaglandins after seizures are associated with consistent manifestation of neuronal damage.