Role of central cannabinoid receptor GPR18 in cardiovascular regulation

The primary goal of this study was to characterize the role of GPR18 in the rostral ventrolateral medulla (RVLM) in blood pressure (BP) regulation and elucidate the mechanisms involved in central GPR18 mediated hypotensive response. The central hypothesis of this study was "central GPR18 signaling exerts a tonic sympathoinhibitory effect". The data provide the first evidence for expression of GPR18 in the RVLM, the cardiovascular regulatory nuclei of the brainstem and its co-localization in tyrosine hydroxylase (TH)-expressing neurons as well as in RVLM neurons expressing cannabinoid� receptors (CB�R). Also intra-RVLM activation of GPR18 receptors with abnormal cannabidiol (Abn CBD) produced a dose-dependent reduction in BP in conscious male Sprague Dawley rats whereas blockade of those receptors with 1, 3-dimethoxy-5-methyl-2-[(1R, 6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl] benzene (O-1918) increased BP and abrogated the Abn CBD-evoked reduction in BP. Additional studies tested the hypothesis that the negligible hypotensive effect caused by the endogenous GPR18 ligand n-arachidonoyl glycine (NAGly) could be due to concurrent activation of CB�R in the RVLM. Our findings supported this hypothesis because NAGly-evoked hypotension was doubled following RVLM CB�R blockade (SR141716). Ex-vivo studies revealed that intra-RVLM GPR18 activation (Abn CBD; 0.4 [mu]g) enhanced RVLM Akt, ERK1/2 and nNOS phosphorylation and increased adiponectin (ADN) levels, during the hypotensive response. In contrast, prior GPR18 receptor blockade (O-1918) produced the opposite effects, and abrogated Abn CBD-evoked responses in conscious Sprague Dawley rats. Inhibition of RVLM PI3K/Akt (wortmannin), ERK1/2 (PD98059) or nNOS (N[omega]-propyl-L-arginine, NPLA) or activation of adenylyl cyclase (forskolin) virtually abolished the intra-RVLM Abn CBD-evoked hypotension. Further, wortmannin, PD98059, NPLA or forskolin abrogated the GPR18-mediated increases in RVLM Akt, ERK1/2, nNOS phosphorylation and ADN levels, along with increased ROS generation. Our in-vitro studies show that GPR18 is co-expressed with CB1R in nPC12 cells. Our in-vitro studies are in line with our in-vivo findings in normotensive rats indicating that GPR18 signals through the PI3K/Akt-ERK1/2-nNOS/ADN pathway. Additionally, our confocal imaging findings show that GPR18 is associated with lipid rafts (LRs). Furthermore, activation of GPR18 (Abn CBD/ NAGly) displaces it from the LRs and this response was abrogated by prior GPR18 blockade (O-1918). Furthermore, cholesterol depletion by methyl-[beta]-cyclodextrin (M[beta]CD) enhanced GPR18 signaling and uncovered O-1918 blockade in nPC12 cells. Collectively, these studies provide insight into identifying the potential signaling pathway(s) triggered by central GPR18 activation in conscious animals and the potential role of lipid rafts in modulating GPR18 signaling in-vitroPh.D

Food allergen extracts to diagnose food-induced allergic diseases

OBJECTIVE: To review the manufacturing procedures of food allergen extracts and applicable regulatory requirements from government agencies, potential approaches to standardization, and clinical application of these products. The effects of thermal processing on allergenicity of common food allergens are also considered. DATA SOURCES: A broad literature review was conducted on the natural history of food allergy, the manufacture of allergen extracts, and the allergenicity of heated food. Regulations, guidance documents, and pharmacopoeias related to food allergen extracts from the United States and Europe were also reviewed. STUDY SELECTIONS: Authoritative and peer-reviewed research articles relevant to the topic were chosen for review. Selected regulations and guidance documents are current and relevant to food allergen extracts. RESULTS: Preparation of a food allergen extract may require careful selection and identification of source materials, grinding, defatting, extraction, clarification, sterilization, and product testing. Although extractions for all products licensed in the United States are performed using raw source materials, many foods are not consumed in their raw form. Heating foods may change their allergenicity, and doing so before extraction may change their allergenicity and the composition of the final product. CONCLUSION: The manufacture of food allergen extracts requires many considerations to achieve the maximal quality of the final product. Allergen extracts for a select number of foods may be inconsistent between manufacturers or unreliable in a clinical setting, indicating a potential area for future improvement

Role of central cannabinoid receptor GPR18 in cardiovascular regulation

The primary goal of this study was to characterize the role of GPR18 in the rostral ventrolateral medulla (RVLM) in blood pressure (BP) regulation and elucidate the mechanisms involved in central GPR18 mediated hypotensive response. The central hypothesis of this study was "central GPR18 signaling exerts a tonic sympathoinhibitory effect". The data provide the first evidence for expression of GPR18 in the RVLM, the cardiovascular regulatory nuclei of the brainstem and its co-localization in tyrosine hydroxylase (TH)-expressing neurons as well as in RVLM neurons expressing cannabinoid1 receptors (CB1R). Also intra-RVLM activation of GPR18 receptors with abnormal cannabidiol (Abn CBD) produced a dose-dependent reduction in BP in conscious male Sprague Dawley rats whereas blockade of those receptors with 1, 3-dimethoxy-5-methyl-2-[(1R, 6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl] benzene (O-1918) increased BP and abrogated the Abn CBD-evoked reduction in BP. Additional studies tested the hypothesis that the negligible hypotensive effect caused by the endogenous GPR18 ligand n-arachidonoyl glycine (NAGly) could be due to concurrent activation of CB1R in the RVLM. Our findings supported this hypothesis because NAGly-evoked hypotension was doubled following RVLM CB1R blockade (SR141716). Ex-vivo studies revealed that intra-RVLM GPR18 activation (Abn CBD\; 0.4 [mu]g) enhanced RVLM Akt, ERK1/2 and nNOS phosphorylation and increased adiponectin (ADN) levels, during the hypotensive response. In contrast, prior GPR18 receptor blockade (O-1918) produced the opposite effects, and abrogated Abn CBD-evoked responses in conscious Sprague Dawley rats. Inhibition of RVLM PI3K/Akt (wortmannin), ERK1/2 (PD98059) or nNOS (N[omega]-propyl-L-arginine, NPLA) or activation of adenylyl cyclase (forskolin) virtually abolished the intra-RVLM Abn CBD-evoked hypotension. Further, wortmannin, PD98059, NPLA or forskolin abrogated the GPR18-mediated increases in RVLM Akt, ERK1/2, nNOS phosphorylation and ADN levels, along with increased ROS generation. Our in-vitro studies show that GPR18 is co-expressed with CB1R in nPC12 cells. Our in-vitro studies are in line with our in-vivo findings in normotensive rats indicating that GPR18 signals through the PI3K/Akt-ERK1/2-nNOS/ADN pathway. Additionally, our confocal imaging findings show that GPR18 is associated with lipid rafts (LRs). Furthermore, activation of GPR18 (Abn CBD/ NAGly) displaces it from the LRs and this response was abrogated by prior GPR18 blockade (O-1918). Furthermore, cholesterol depletion by methyl-[beta]-cyclodextrin (M[beta]CD) enhanced GPR18 signaling and uncovered O-1918 blockade in nPC12 cells. Collectively, these studies provide insight into identifying the potential signaling pathway(s) triggered by central GPR18 activation in conscious animals and the potential role of lipid rafts in modulating GPR18 signaling in-vitr