Insights for New and Developing Rural Family Medicine Residency Programs.

PURPOSE: Rural family medicine residency programs (RFMRPs) encounter unique hardships that threaten their sustainability and efficacy despite their recent success at addressing the rural physician shortage. The aim of this study was to explore strategies employed by RFMRP program directors from across the United States to strengthen their programs in the context of evolving paradigms in graduate medical education (GME). METHODS: The authors conducted a qualitative semistructured telephone interview with 19 program directors of RFMRPs in June and July of 2020. Interviews were audio-recorded, transcribed verbatim, and analyzed using thematic content analysis. FINDINGS: Two major themes emerged: (1) community enrichment and (2) the ability to evolve to meet demands. Community enrichment had five subthemes: evaluate local resources, prioritize community buy-in, design a robust continuity clinic, identify or cultivate a local physician champion, and support faculty and physician preceptors. Programs evolving to meet demands had four subthemes: frequently revisit program mission to align with scope of family medicine, redefine expectations in medical education, integrate longitudinal experiences, and implement innovation in curriculum design. CONCLUSIONS: Community enrichment and programs' ability to evolve to meet demands are important attributes of a successful RFMRP. Our findings highlight strategies utilized by RFMRPs to help meet the needs of the changing landscape of rural family medicine GME and help identify best practices for developing RFMRPs.

An outcomes research perspective on medical education: Has anything changed in the last 18 years?

PURPOSE: Medical education research focused on patient-centered outcomes holds the promise of improved decision-making by medical educators. In 2001, Prystowsky and Bordage demonstrated that patient-centered outcomes were evaluated in fewer than one percent of studies published in a survey of major medical education journals. Though many have called for increased inclusion of patient-centered outcomes in medical education literature, it remains uncertain to what degree this need has been addressed systematically. METHODS: Using the same data sources as in the original report (Academic Medicine, Medical Education, and Teaching and Learning in Medicine), we sought to replicate Prystowsky and Bordage's study. We extracted data from original empirical research reports from these three journal sources for the years 2014-2016. We selected 652 articles that met the inclusion criteria for further analysis. RESULTS: Study participants were largely trainees (64% of studies) or faculty (25% of studies). Only 2% of studies included patients as active or passive participants. Study outcomes reported were satisfaction (40% of studies), performance (39%), professionalism (20%), and cost (1%). CONCLUSIONS: These results do not differ significantly from the original 2001 study. The medical education literature as represented in these three prominent journals has made little progress in placing a greater focus on patient-centered outcomes.

An innovative pharmacology curriculum for medical students: promoting higher order cognition, learner-centered coaching, and constructive feedback through a social pedagogy framework.

BACKGROUND: Ongoing developments in medical education recognize the move to curricula that support self-regulated learning processes, skills of thinking, and the ability to adapt and navigate uncertain situations as much as the knowledge base of learners. Difficulties encountered in pursuing this reform, especially for pharmacology, include the tendency of beginner learners not to ask higher-order questions and the potential incongruency between creating authentic spaces for self-directed learning and providing external expert guidance. We tested the feasibility of developing, implementing, and sustaining an innovative model of social pedagogy as a strategy to address these challenges. METHODS: Constructivism, communities of practice, and networked learning theory were selected as lenses for development of the model. Three hundred sixty-five first-year medical students participated between 2014 and 2018; they were introduced to pharmacodynamics and pharmacokinetics via 15 online modules that each included: learning objectives, a clinical vignette, teaching video, cumulative concept map, and small group wiki assignment. Five-person communities organized around the 15 wiki assignments were a key component where learners answered asynchronous, case-based questions that touched iteratively on Bloom's cognitive taxonomy levels. The social pedagogy model's wiki assignments were explored using abductive qualitative data analysis. RESULTS: Qualitative analysis revealed that learners acquired and applied a conceptual framework for approaching pharmacology as a discipline, and demonstrated adaptive mastery by evaluating and interacting competently with unfamiliar drug information. Learners and faculty acquired habits of self-directed assessment seeking and learner-centered coaching, respectively; specifically, the model taught learners to look outward to peers, faculty, and external sources of information for credible and constructive feedback, and that this feedback could be trusted as a basis to direct performance improvement. 82-94% of learners rated the social pedagogy-based curriculum valuable. CONCLUSIONS: This social pedagogy model is agnostic with regard to pharmacology and type of health professional learner; therefore, we anticipate its benefits to be transferable to other disciplines.

A Case Series: Successfully Preventing COVID-19 Outbreak in a Residential Community Setting at a Drug and Alcohol Addiction Treatment Center.

Coronavirus disease 2019 (COVID-19) has reduced the capacity of many addiction treatment centers, limiting access to safe, continual treatment for people with substance use disorders (SUD) in the setting of a pandemic. Here, we describe the COVID-19 screening process of a residential addiction treatment center in rural Connecticut that has had no outbreaks, closures, or reductions in capacity since the pandemic began. Out of 420 patients screened for COVID-19 from 1 February to 1 July, five patients tested positive for COVID-19: four prior to entering its residential community setting, and one after entering the residential community, resulting in no COVID-19 spread to other patients. Patient 1 presented from home and tested positive during screening prior to entry into the community. The primary care provider for patient 2 notified staff of a recent pos-itive COVID-19 test prior to the patient's arrival on-site. Patient 3 had a COVID-19 infection in the weeks prior to arrival and tested positive during initial screening. Patient 4 tested positive af-ter coming from another addiction treatment facility that was shut down due to a COVID-19 outbreak. Patient 5 tested negative for COVID-19 during initial screening, entered the residential community, and later tested positive. It is imperative that in-person support for SUD continues during the pandemic. This case report highlights the importance of implementing a variety of tools in an effective screening process, including polymerase chain reaction screening and daily symptomology and temperature screening, which may help prevent further closures or reductions in capacity of addiction treatment centers during the COVID-19 pandemic or future outbreaks.

Contextualizing adolescents' self-awareness of problematic mobile phone use: a preliminary study.

Adolescents engage cognitively, emotionally, and behaviorally with smartphones. Growing evidence suggests they struggle to interact with them in moderation, which has been framed in relation to behavioral addiction as problematic mobile phone use. This study contextualized 13-15 year-old adolescents' self-awareness of problematic mobile phone use. Focus groups were conducted with 11 adolescents who assessed themselves using the problematic use of mobile phones scale. The authors used interpretative phenomenological epistemology as a guiding framework. Audio recordings were analyzed qualitatively using a constant comparison approach. Students agreed or strongly agreed with multiple dimensions of the problematic mobile phone use construct. Four major themes emerged in relation to circumstances, factors, processes, constraints, and opportunities: drivers of excessive smartphone use, with family or friends, barriers to healthier smartphone use, and nighttime habits. Adolescents' assessment of perceived proper versus problematic mobile phone can inform hypotheses targeted at improving overall wellness and developing healthy habits in adolescence that carry over into young adulthood and beyond.

Twelve tips for developing and implementing curriculum in dedicated 'collaborative classrooms'.

Many health professional schools may be investing time and resources on dedicated educational spaces intended to promote collaborative learning. Alone, innovative physical space or technologies are not sufficient to ensure success in this. Lesson plans informed by collaborative praxis, individual motivation, faculty development, learner feedback, and team interactions also play a necessary and substantial role. We have used faculty observations, quantitative and qualitative student evaluation data, and the existing educational literature to provide twelve tips on leveraging curricular content, activity setup, physical space, learner behavior, and faculty facilitation to make the most of collaborative learning spaces.

Evidence for a GPR18 Role in Chemotaxis, Proliferation, and the Course of Wound Closure in the Cornea.

PURPOSE: We previously showed that cannabinoid-related GPR18 receptors are present in the murine corneal epithelium, but their function remains unknown. The related CB1 receptors regulate corneal healing, possibly via chemotaxis. We therefore examined a potential role for GPR18 in corneal epithelial chemotaxis and wound healing. METHODS: We examined GPR18 messenger RNA (mRNA) and protein expression in the cornea. We additionally examined GPR18 action in cultured bovine corneal epithelial cells (bCECs) using Boyden and tracking assays, as well as proliferation and signaling. Finally, we examined wound closure in murine corneal explants. RESULTS: GPR18 mRNA was upregulated with injury in the mouse cornea. GPR18 protein was present in basal epithelial cells of the mouse and cow and redistributed to the wound site upon injury. GPR18 ligand N-arachidonoylglycine induced bCEC chemotaxis. The endocannabinoid arachidonoylethanolamine also induced chemotaxis via fatty acid amide hydrolase-mediated metabolism to N-arachidonoylglycine. GPR18 receptor activation additionally induced bCEC proliferation. In an explant model, the GPR18 antagonist O-1918 slowed corneal epithelial cell migration and the rate of corneal wound closure. CONCLUSIONS: Corneal GPR18 activation induced both chemotaxis and proliferation in corneal epithelial cells in vitro and impacted wound healing. GPR18 may contribute to the maintenance of corneal integrity.

Nutrition as Medicine, Food Sensitivities, and Chronic Disease: a Mini Curriculum Shaped by Transformative Learning Theory.

Nutrition-related diseases are preventable twenty-first-century health problems. Students report being underprepared for nutritional therapeutics. We developed a mini, spiral curriculum shaped by transformative learning theory and centered on nutrition as medicine, food sensitivities, and chronic disease to kick-start a shift in cognition, attitudes, and skills. Seven demographically representative students participated in the 3-week curriculum. Visual, textual, and verbal data were captured and analyzed qualitatively. We found the curriculum evoked paradigm transformation that persisted 6 months later and facilitated opportunities for our cohort to self-identify learning gaps and consider how they related to their current and future goals for patient care.

Review article: Idle 'just-in-case' peripheral intravenous cannulas in the emergency department: Is something wrong?

Peripheral intravenous cannula (PIVC) placement is often an essential emergency medicine precursor to lifesaving treatment, but it is not harmless. Patients frequently and without proper consideration of the consequences receive a 'just-in-case' PIVCs as part of their assessment and admission, which, in a not insignificant number of patients, remains unused or idle in situ. We reviewed the literature and performed a thematic analysis of data collated from 21 articles published in the past 24 years regarding redundant PIVCs. The following five common themes emerged: heterogeneous prevalence data on post-insertion PIVC usage, preventable intravascular complications, financial burden, loss of time and a culture of over-investigating. The prevalence of PIVC insertions and idle PIVCs was heterogeneous among these publications; the median ED idle PIVC prevalence value was 32.4%. This practice is associated with compromised patient safety, squandered finances and misdirected practitioner time. Cultures of convenience and shortfalls in PIVC-related education facilitate the prevalence of idle PIVCs.

Cannabinoid-induced chemotaxis in bovine corneal epithelial cells.

PURPOSE: Cannabinoid CB1 receptors are found in abundance in the vertebrate eye, with most tissue types expressing this receptor. However, the function of CB1 receptors in corneal epithelial cells (CECs) is poorly understood. Interestingly, the corneas of CB1 knockout mice heal more slowly after injury via a mechanism proposed to involve protein kinase B (Akt) activation, chemokinesis, and cell proliferation. The current study examined the role of cannabinoids in CEC migration in greater detail. METHODS: We determined the role of CB1 receptors in corneal healing. We examined the consequences of their activation on migration and proliferation in bovine CECs (bCECs). We additionally examined the mRNA profile of cannabinoid-related genes and CB1 protein expression as well as CB1 signaling in bovine CECs. RESULTS: We now report that activation of CB1 with physiologically relevant concentrations of the synthetic agonist WIN55212-2 (WIN) induces bCEC migration via chemotaxis, an effect fully blocked by the CB1 receptor antagonist SR141716. The endogenous agonist 2-arachidonoylglycerol (2-AG) also enhances migration. Separately, mRNA for most cannabinoid-related proteins are present in bovine corneal epithelium and cultured bCECs. Notably absent are CB2 receptors and the 2-AG synthesizing enzyme diglycerol lipase-α (DAGLα). The signaling profile of CB1 activation is complex, with inactivation of mitogen-activated protein kinase (MAPK). Lastly, CB1 activation does not induce bCEC proliferation, but may instead antagonize EGF-induced proliferation. CONCLUSIONS: In summary, we find that CB1-based signaling machinery is present in bovine cornea and that activation of this system induces chemotaxis.

Novel endogenous N-acyl amides activate TRPV1-4 receptors, BV-2 microglia, and are regulated in brain in an acute model of inflammation.

A family of endogenous lipids, structurally analogous to the endogenous cannabinoid, N-arachidonoyl ethanolamine (Anandamide), and called N-acyl amides have emerged as a family of biologically active compounds at TRP receptors. N-acyl amides are constructed from an acyl group and an amine via an amide bond. This same structure can be modified by changing either the fatty acid or the amide to form potentially hundreds of lipids. More than 70 N-acyl amides have been identified in nature. We have ongoing studies aimed at isolating and characterizing additional members of the family of N-acyl amides in both central and peripheral tissues in mammalian systems. Here, using a unique in-house library of over 70 N-acyl amides we tested the following three hypotheses: (1) Additional N-acyl amides will have activity at TRPV1-4, (2) Acute peripheral injury will drive changes in CNS levels of N-acyl amides, and (3) N-acyl amides will regulate calcium in CNS-derived microglia. Through these studies, we have identified 20 novel N-acyl amides that collectively activate (stimulating or inhibiting) TRPV1-4. Using lipid extraction and HPLC coupled to tandem mass spectrometry we showed that levels of at least 10 of these N-acyl amides that activate TRPVs are regulated in brain after intraplantar carrageenan injection. We then screened the BV2 microglial cell line for activity with this N-acyl amide library and found overlap with TRPV receptor activity as well as additional activators of calcium mobilization from these lipids. Together these data provide new insight into the family of N-acyl amides and their roles as signaling molecules at ion channels, in microglia, and in the brain in the context of inflammation.

Δ(9)-THC and N-arachidonoyl glycine regulate BV-2 microglial morphology and cytokine release plasticity: implications for signaling at GPR18.

Microglial cells are extremely plastic and undergo a variety of CNS-prompted shape changes relative to their location and current role. Signaling molecules from neurons also regulate microglial cytokine production. Neurons are known to employ the endogenous cannabinoid system to communicate with other cells of the CNS. N-arachidonoyl glycine (NAGly) and Δ(9)-tetrahydrocannabinol (Δ(9)-THC) signaling via GPR18 has been introduced as an important new target in microglial-neuronal communication. Our hypothesis is that endogenous NAGly-GPR18 signaling regulates phenotypic shape and cytokine production in microglia, and is mimicked by Δ(9)-THC in the BV-2 microglia model system. BV-2 microglia were exposed to NAGly and Δ(9)-THC or Vh for 12 h, which resulted in significant differences in the cell morphologies expressed. Cannabidiol (CBD) was effective at antagonizing the effects of both NAGly and Δ(9)-THC. Using ELISA-based microarrays, BV-2 microglia were exposed to NAGly and Δ(9)-THC or Vh for 3 h and the presence of 40 cytokines in the culture media quantified. Production of Axl, CD40, IGF-I, OPN, and Pro-MMP-9 were significantly altered by NAGly and Δ(9)-THC, and antagonized by CBD. These data add to an emerging profile that emphasizes NAGly as a component of an endogenous system present in the CNS that tightly integrates microglial proliferation, recruitment, and adhesion with neuron-glia interactivity and tissue remodeling.

siRNA knockdown of GPR18 receptors in BV-2 microglia attenuates N-arachidonoyl glycine-induced cell migration.

BACKGROUND: Neurons are known to employ the endogenous cannabinoid system to communicate with other cells of the CNS. Endocannabioid signaling recruits microglia toward neurons by engaging cannabinoid CB2 and abnormal cannabidiol (Abn-CBD) receptors. The Abn-CBD receptor is a prominent atypical cannabinoid receptor that had been discriminated by means of various pharmacological and genetic tools but remained to be identified at the molecular level. We recently introduced N-arachidonoyl glycine (NAGly) signaling via GPR18 receptors as an important novel signaling mechanism in microglial-neuronal communication. NAGly is an endogenous, enzymatically oxygenated metabolite of the endocannabinoid N-arachidonoyl ethanolamide (AEA). Our recent studies support strongly two hypotheses; first that NAGly initiates directed microglial migration in the CNS through activation of GPR18, and second that GPR18 is the Abn-CBD receptor. Here we present siRNA knockdown data in further support of these hypotheses. FINDINGS: A GPR18-targetting siRNA pSUPER G418 GFP cDNA plasmid was created and transfected into BV-2 microglia. Successfully transfected GFP+ GPR18 siRNA BV-2 microglia displayed reduced GPR18 mRNA levels and immunocytochemical staining. Cell migration induced by 1 µM concentrations of NAGly, O-1602 and Abn-CBD were significantly attenuated in GFP+ cells. CONCLUSIONS: Our data provide definitive evidence that these compounds, characteristic of Abn-CBD receptor pharmacology, are acting via GPR18 in BV-2 microglia. A fuller understanding of the hitherto unidentified cannabinoid receptors such as GPR18; their molecular interactions with endogenous ligands; and how phytocannabinoids influence their signaling is vital if we are to comprehensively assess the function of the endogenous cannabinoid signaling system in human health and disease.

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.

The atypical cannabinoid O-1602 protects against experimental colitis and inhibits neutrophil recruitment.

BACKGROUND: Cannabinoids are known to reduce intestinal inflammation. Atypical cannabinoids produce pharmacological effects via unidentified targets. We were interested in whether the atypical cannabinoid O-1602, reportedly an agonist of the putative cannabinoid receptor GPR55, reduces disease severity of dextran sulfate sodium (DSS) and trinitrobenzene sulfonic acid (TNBS)-induced colitis in C57BL/6N and CD1 mice. METHODS: DSS (2.5% and 4%) was supplied in drinking water for 1 week while TNBS (4 mg) was applied as a single intrarectal bolus. RESULTS: Both treatments caused severe colitis. Injection of O-1602 (5 mg/kg intraperitoneally) significantly reduced macroscopic and histological colitis scores, and myeloperoxidase activity. The protective effect was still present in cannabinoid receptor 1 (CB1) and 2 (CB2) double knockout mice and mice lacking the GPR55 gene. To investigate a potential mechanism underlying the protection by O-1602 we performed neutrophil chemotactic assays. O-1602 concentration-dependently inhibited migration of murine neutrophils to keratinocyte-derived chemokine (KC), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the N-formyl-peptide receptor ligand WKYMVm. The inhibitory effect of O-1602 was preserved in neutrophils from CB1/CB2 double knockout and GPR55 knockout mice. No differences were seen in locomotor activity between O-1602-treated and control mice, indicating lack of central sedation by this compound. CONCLUSIONS: Our data demonstrate that O-1602 is protective against experimentally induced colitis and inhibits neutrophil recruitment independently of CB1, CB2, and GPR55 receptors. Thus, atypical cannabinoids represent a novel class of therapeutics that may be useful for the treatment of inflammatory bowel diseases.

N-arachidonoyl glycine, an abundant endogenous lipid, potently drives directed cellular migration through GPR18, the putative abnormal cannabidiol receptor.

BACKGROUND: Microglia provide continuous immune surveillance of the CNS and upon activation rapidly change phenotype to express receptors that respond to chemoattractants during CNS damage or infection. These activated microglia undergo directed migration towards affected tissue. Importantly, the molecular species of chemoattractant encountered determines if microglia respond with pro- or anti-inflammatory behaviour, yet the signaling molecules that trigger migration remain poorly understood. The endogenous cannabinoid system regulates microglial migration via CB2 receptors and an as yet unidentified GPCR termed the 'abnormal cannabidiol' (Abn-CBD) receptor. Abn-CBD is a synthetic isomer of the phytocannabinoid cannabidiol (CBD) and is inactive at CB1 or CB2 receptors, but functions as a selective agonist at this Gi/o-coupled GPCR. N-arachidonoyl glycine (NAGly) is an endogenous metabolite of the endocannabinoid anandamide and acts as an efficacious agonist at GPR18. Here, we investigate the relationship between NAGly, Abn-CBD, the unidentified 'Abn-CBD' receptor, GPR18, and BV-2 microglial migration. RESULTS: Using Boyden chamber migration experiments, yellow tetrazolium (MTT) conversion, In-cell Western, qPCR and immunocytochemistry we show that NAGly, at sub-nanomolar concentrations, and Abn-CBD potently drive cellular migration in both BV-2 microglia and HEK293-GPR18 transfected cells, but neither induce migration in HEK-GPR55 or non-transfected HEK293 wildtype cells. Migration effects are blocked or attenuated in both systems by the 'Abn-CBD' receptor antagonist O-1918, and low efficacy agonists N-arachidonoyl-serine and cannabidiol. NAGly promotes proliferation and activation of MAP kinases in BV-2 microglia and HEK293-GPR18 cells at low nanomolar concentrations - cellular responses correlated with microglial migration. Additionally, BV-2 cells show GPR18 immunocytochemical staining and abundant GPR18 mRNA. qPCR demonstrates that primary microglia, likewise, express abundant amounts of GPR18 mRNA. CONCLUSIONS: NAGly is the most effective lipid recruiter of BV-2 microglia currently reported and its effects mimic those of Abn-CBD. The data generated from this study supports the hypothesis that GPR18 is the previously unidentified 'Abn-CBD' receptor. The marked potency of NAGly acting on GPR18 to elicit directed migration, proliferation and perhaps other MAPK-dependent phenomena advances our understanding of the lipid-based signaling mechanisms employed by the CNS to actively recruit microglia to sites of interest. It offers a novel research avenue for developing therapeutics to elicit a self-renewing population of neuroregenerative microglia, or alternatively, to prevent the accumulation of misdirected, pro-inflammatory microglia which contribute to and exacerbate neurodegenerative disease.

Endogenous cannabinoids and neutrophil chemotaxis.

Neutrophils are the earliest inflammatory cell to infiltrate tissue, playing an important role in early phagocytosis. Under pathological conditions, pro-inflammatory actions of neutrophils contribute to the development of various inflammatory diseases. G(i) protein-coupled cell-surface receptors are an essential component of pro-migratory responses in leukocytes; however, few investigations regarding inhibitors of cell migration have been reported. Kurihara et al. (2006) and McHugh et al. (2008) have revealed that certain endogenous cannabinoids and lipids are potent inhibitors of induced human neutrophil migration. McHugh et al. implicate a novel SR141716A-sensitive pharmacological target distinct from cannabinoid CB(1) and CB(2) receptors, which is antagonized by N-arachidonoyl-l-serine; and that the CB(2) receptor exerts negative co-operativity upon this receptor. Kurihara et al. demonstrate that fMLP-induced RhoA activity is decreased following endocannabinoid pretreatment, disrupting the front/rear polarization necessary for neutrophils to engage in chemotaxis. The therapeutic potential of exploiting endocannabinoids as neutrophilic chemorepellants is plain to see.

Orphan endogenous lipids and orphan GPCRs: a good match.

A large and growing family of over 70 endogenous lipids of the basic structure N-acyl amide has been identified during the last 10 years. Only a few of these lipids have been characterized for biological activity, however, those that have shown a wide-range of activity may act at G-protein coupled receptors (GPCRs). Like orphan GPCRs that are identified as being in the genome and expressed in tissue, the majority of these endogenous lipids many produced throughout the body, some predominately in nervous tissue, remain orphaned. Here, we give a brief history of these orphan lipids and highlight the activity of N-arachidonoyl glycine, and farnesyl pyrophosphate at the orphan receptors GPR18 and GPR92, respectively, as well as summarizing the biological and pharmacological data for the recently identified N-palmitoyl glycine that suggests activity at a novel GPCR. Working to deorphanize both lipids and GPCRs together provides a unique opportunity for a greater understanding of cellular signaling and a challenge to find them all a home.

N-palmitoyl glycine, a novel endogenous lipid that acts as a modulator of calcium influx and nitric oxide production in sensory neurons.

N-arachidonoyl glycine is an endogenous arachidonoyl amide that activates the orphan G protein-coupled receptor (GPCR) GPR18 in a pertussis toxin (PTX)-sensitive manner and produces antinociceptive and antiinflammatory effects. It is produced by direct conjugation of arachidonic acid to glycine and by oxidative metabolism of the endocannabinoid anandamide. Based on the presence of enzymes that conjugate fatty acids with glycine and the high abundance of palmitic acid in the brain, we hypothesized the endogenous formation of the saturated N-acyl amide N-palmitoyl glycine (PalGly). PalGly was partially purified from rat lipid extracts and identified using nano-high-performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry. Here, we show that PalGly is produced after cellular stimulation and that it occurs in high levels in rat skin and spinal cord. PalGly was up-regulated in fatty acid amide hydrolase knockout mice, suggesting a pathway for enzymatic regulation. PalGly potently inhibited heat-evoked firing of nociceptive neurons in rat dorsal horn. In addition, PalGly induced transient calcium influx in native adult dorsal root ganglion (DRG) cells and a DRG-like cell line (F-11). The effect of PalGly on the latter cells was characterized by strict structural requirements, PTX sensitivity, and dependence on the presence of extracellular calcium. PalGly-induced calcium influx was blocked by the nonselective calcium channel blockers ruthenium red, 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SK&F96365), and La3+. Furthermore, PalGly contributed to the production of NO through calcium-sensitive nitric-oxide synthase enzymes present in F-11 cells and was inhibited by the nitric-oxide synthase inhibitor 7-nitroindazole.

Inhibition of human neutrophil chemotaxis by endogenous cannabinoids and phytocannabinoids: evidence for a site distinct from CB1 and CB2.

Here, we show a novel pharmacology for inhibition of human neutrophil migration by endocannabinoids, phytocannabinoids, and related compounds. The endocannabinoids virodhamine and N-arachidonoyl dopamine are potent inhibitors of N-formyl-l-methionyl-l-leucyl-l-phenylalanine-induced migration of human neutrophils, with IC(50) values of 0.2 and 8.80 nM, respectively. The endocannabinoid anandamide inhibits human neutrophil migration at nanomolar concentrations in a biphasic manner. The phytocannabinoid (-)-cannabidiol is a partial agonist, being approximately 40 fold more potent than (+)-cannabidiol; abnormal-cannabidiol is a full agonist. Furthermore, the abnormal-cannabidiol (CBD) analog trans-4-[3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-methyl-1,3-benzenediol (O-1602) inhibits migration, with an IC(50) value of 33 nM. This reported profile of agonist efficacy and potency parallels with the pharmacology of the novel "abnormal-cannabidiol" receptor or a related orphan G protein-coupled receptor, which are already known to modulate cell migration. Although having no effect alone, N-arachidonoyl l-serine attenuated inhibition of human neutrophil migration induced by anandamide, virodhamine, and abnormal-CBD. Our data also suggest that there is cross-talk/negative co-operativity between the cannabinoid CB(2) receptor and this novel target: CB(2) receptor antagonists significantly enhance the inhibition observed with anandamide and virodhamine. This study reveals that certain endogenous lipids, phytocannabinoids, and related ligands are potent inhibitors of human neutrophil migration, and it implicates a novel pharmacological target distinct from cannabinoid CB(1) and CB(2) receptors; this target is antagonized by the endogenous compound N-arachidonoyl l-serine. Furthermore, our findings have implications for the potential pharmacological manipulation of elements of the endocannabinoid system for the treatment of various inflammatory conditions.