Cannabinoid Receptor Protein Research Articles

Long-term Effects of Cannabidiol and/or Fentanyl Exposure in Rats Submitted to Neonatal Pain

The current study aimed to evaluate anxiety behavior, hippocampal ionized calcium-binding adaptor molecule 1 (Iba1) and cannabinoid receptor 1 (CB1) gene expression, and nociceptive response in adulthood after a combination of fentanyl and cannabidiol (CBD) for nociceptive stimuli induced during the first week of life in rats. Complete Freund’s adjuvant-induced inflammatory nociceptive insult on postnatal day (PN) 1 and PN3. Both fentanyl and CBD were used alone or in combination from PN1 to PN7. Behavioral and nociceptive tests were performed at PN60 and PN62. The expression of the microglial calcium-binding proteins Iba1 and CB1 was detected in the hippocampus using reverse Quantitative polymerase chain reaction (qPCR) and immunohistochemistry. Our results suggest that the anxiety behavior response and immune activation in adult life depend on the CBD dose combined with fentanyl for the nociceptive stimuli induced during the first week of life. Treatment of neonatal nociceptive insult with CBD and opioids showed significant dose-dependent and male-female differences. The increased gene expression in the hippocampus of the analyzed cannabinoid gene supports this data. In addition, treatment with fentanyl led to an increase in CB1 protein expression. Moreover, the expression of Iba1 varied according to the administered dose of CBD and may or may not be associated with the opioid. A lower dose of CBD during the inflammatory period was associated with enhanced anxiety in adult life. PerspectiveThe treatment of nociceptive stimuli with CBD and opioids during the first week of life demonstrated significant sex differences in adult life on anxiety behavior and supraspinal pain sensitivity.

α-Dystrobrevin knockout mice have increased motivation for appetitive reward and altered brain cannabinoid receptor 1 expression

α-Dystrobrevin (α-DB) is a major component of the dystrophin-associated protein complex (DAPC). Knockout (KO) of α-DB in the brain is associated with astrocytic abnormalities and loss of neuronal GABA receptor clustering. Mutations in DAPC proteins are associated with altered dopamine signaling and cognitive and psychiatric disorders, including schizophrenia. This study tested the hypothesis that motivation and associated underlying biological pathways are altered in the absence of α-DB expression. Male wildtype and α-DB KO mice were tested for measures of motivation, executive function and extinction in the rodent touchscreen apparatus. Subsequently, brain tissues were evaluated for mRNA and/or protein levels of dysbindin-1, dopamine transporter and receptor 1 and 2, mu opioid receptor 1 (mOR1) and cannabinoid receptor 1 (CB1). α-DB KO mice had significantly increased motivation for the appetitive reward, while measures of executive function and extinction were unaffected. No differences were observed between wildtype and KO animals on mRNA levels of dysbindin-1 or any of the dopamine markers. mRNA levels of mOR1were significantly decreased in the caudate-putamen and nucleus accumbens of α-DB KO compared to WT animals, but protein levels were unaltered. However, CB1 protein levels were significantly increased in the prefrontal cortex and decreased in the nucleus accumbens of α-DB KO mice. Triple-labelling immunohistochemistry confirmed that changes in CB1 were not specific to astrocytes. These results highlight a novel role for α-DB in the regulation of appetitive motivation that may have implications for other behaviours that involve the dopaminergic and endocannabinoid systems.

Possible Association of Nucleobindin-1 Protein with Depressive Disorder in Patients with HIV Infection.

Background: Mental disorders linked with dysfunction in the temporal cortex, such as anxiety and depression, can increase the morbidity and mortality of people living with HIV (PLWHA). Expressions of both nucleobindin 1 (NUCB1) and cannabinoid receptor 1 (CNR1) in the neurons have been found to alter in patients with depressive disorder, but whether it is involved in the development of depression in the context of HIV infection is unknown. Objectives To investigate the effects of NUCB1 on depressive disorder among PLWHA and preliminarily explore the underlying molecular mechanisms. Methods: Individuals who were newly HIV diagnosed were assessed on the Hospital Anxiety and Depression scale (HADS). Then SHIV-infected rhesus monkeys were used to investigate the possible involvement of the NUCB1 and the CNR1 protein in depression-like behavior. Results: The prevalence rate of depression among PLWHA was 27.33% (41/150). The mechanism results showing elevated NUCB1 levels in cerebrospinal fluid from HIV-infected patients suffering from depression were confirmed compared to those of HIV-infected patients. Moreover, the immunohistochemical analysis indicated the expression of NUCB1 in the temporal cortex neurons of SHIV-infected monkeys was higher than that of the healthy control. Conversely, CNR1 expression was down-regulated at protein levels. Conclusions: Depression symptoms are common among PLWHA and associate with NUCB1 expression increases, and NUCB1 may be a potential target for depression.

Endogenous Cannabinoid Receptors Modulate Plasticity at Immature Synapses

To explore the mechanism of endocannabinoid cannabinoid receptor 1 (CB1) receptor pathway that regulates synaptic plasticity in the dorsal horn of the spinal cord of rats with neuropathic pain at different ages. Neonatal, juvenile, and adult male sprague dawley (SD) rats were divided into the spinal nerve preservation injury (SNI), SNI + Anandamide (AEA), SNI + D-AP5, SNI + CNQX, SNI + D-AP5 + AEA, SNI + CNQX + AEA, sham SNI, sham SNI + AEA, sham SNI + D-AP5, sham SNI + CNQX, sham SNI + D-AP5 + AEA, and sham SNI + CNQX + AEA groups, respectively. Paw withdrawal threshold (PWT) and long-term potentiation (LTP) of the spinal dorsal horn PS (field potential) were assessed to judge the spinal cord's functional state. Immunohistochemical staining and Western blot were conducted to detect CB1 protein levels in the spinal dorsal horn. The LTP response in the spinal cord was alleviated in the SNI + AEA group. After treatment with the N-methyl-D-aspartate (NMDA) receptor blocker D-AP5, the LTP of neonatal A nerve was relieved further. After treatment with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blocker CNQX, LTP change in the A nerve was not obvious. The LTP of the A and C nerves were relieved after D-AP5 or CNQX treatment in young and adult animals; however, the blocking effect of CNQX was obvious. The altered levels of PWT and CB1 support these results. The CB1 receptor activation produces analgesia in neonatal rats through NMDA receptor formation for PS inhibitory activity. In juvenile and adult rats, this phenomenon was effectuated through NMDA and AMPA receptors. This difference could be attributed to the varied number of NMDA and/or AMPA receptors activated during development and changes in the NMDA/AMPA receptor ratio.

Intravenous self-administration of delta-9-THC in adolescent rats produces long-lasting alterations in behavior and receptor protein expression.

Rationale:Initial exposure to cannabinoids, including Δ-9-tetrahydrocannabinol (THC), often occurs during adolescence. Considerable neurodevelopmental alterations occur throughout adolescence, and the environmental insult posed by exogenous cannabinoid exposure may alter natural developmental trajectories. Multiple studies suggest that long-lasting deficits in cognitive function occur as a result of adolescent cannabis use, but considerable variability exists in the magnitude of these effects.Objectives:We sought to establish a novel procedure for achieving intravenous THC self-administration in adolescent rats in order to determine if volitional THC intake in adolescence produced indices of addiction-related behavior, altered working memory performance in adulthood, or altered the expression of proteins associated with these behaviors across several brain regions.Methods:Male and female adolescent rats learned to operantly self-administer escalating doses of THC intravenously from PD 32–51. Upon reaching adulthood they were tested in abstinence for cued reinstatement of THC-seeking and working memory performance on a delayed-match-to-sample task. In a separate cohort, glutamatergic, GABAergic, and cannabinoid receptor protein expression was measured in multiple brain regions.Results:Both male and female adolescents self-administered THC and exhibited cue-induced lever pressing throughout abstinence. THC exposed males exhibited slightly enhanced working memory performance in adulthood, and better performance positively correlated with total THC self-administered during adolescence. Adolescent THC-exposed rats exhibited reductions in CB1, GABA and glutamate receptor protein, primarily in the prefrontal cortex, dorsal hippocampus, and ventral tegmental area.Conclusions:These results suggest that THC exposure at self-administered doses can produce moderate behavioral and molecular alterations, including sex-dependent effects on working memory performance in adulthood.

Maternal high-fat diet up-regulates type-1 cannabinoid receptor with estrogen signaling changes in a sex- and depot- specific manner in white adipose tissue of adult rat offspring.

Obesity and high-fat (HF) diet are associated with over activation of the endocannabinoid system (ECS). We have demonstrated that maternal HF diet induces early obesity and modulates cannabinoid signaling in visceral (VIS) and subcutaneous (SUB) white adipose tissue (WAT) in weanling rat offspring. We hypothesized that perinatal maternal HF diet would program the expression of ECS in adipose tissue in a long-term way in parallel to alterations in epigenetic markers and sex hormone signaling. Progenitor female rats received control diet (C, 9% fat) or isocaloric high-fat diet (HF, 28% fat) for 8weeks before mating, gestation, and lactation. All pups were weaned to C diet and they were euthanized at 180days old. Maternal HF diet induced overweight and increased SUB WAT mass of male and female adult offspring. Maternal HF diet induced hypertrophy of VIS and SUB adipocytes only in female offspring associated with increased type 1 cannabinoid receptor protein (CB1) and mRNA (Cnr1) levels. These changes were associated with increased estrogen receptor α binding to Cnr1 promoter in SUB WAT of adult female offspring, which may contribute to higher expression of Cnr1. Increased CB1 signaling in adipose tissue might contribute to higher adiposity programmed by maternal HF diet because endocannabinoids stimulate the accumulation of fat in the adipose tissue. Our findings provide molecular insights into sex-specific targets for anti-obesity therapies based on the endocannabinoid system.

Electroacupuncture alleviates morphine‑induced hyperalgesia by regulating spinal CB1�receptors and ERK1/2 activity

Electroacupuncture (EA), a traditional Chinese therapeutic technique, is considered an effective method for treating certain painful neuropathies induced by various neuropathological damage. The current study investigated the effect of EA on intrathecal (IT) morphine-induced hyperalgesia (MIH) and examined the hypothesis that activation of cannabinoid receptor 1 (CB1) could enhance the antinociceptive effect of EA on MIH via regulation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. Using a rat model of IT MIH, mechanical and thermal hyperalgesia were evaluated by an electronic von Frey filament and hotplate at baseline (1 day before IT administration) and at days 1, 3, 5 and 7 after IT administration. Rats received IT normal saline, IT morphine or IT morphine + EA at ST36-GB34. The protein levels of ERK1/2, phosphorylated (p)-ERK1/2 and CB1 in the spinal cord were assayed by western blotting. Furthermore, the effect of IT injection of the CB1 agonist WIN 55,212-2 and the CB1 antagonist SR141716 on the antinociceptive effect of EA in rats with MIH was investigated. Nociceptive behavior and ERK1/2, phosphorylated (p)-ERK1/2 and CB1 protein levels were evaluated as mentioned above. The results revealed that chronic IT injections of morphine induced a significant decrease in mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) accompanied with remarkable upregulation of p-ERK1/2 in the spinal cord, which could be attenuated by EA at the ST36-GB34 acupoints. In the rat model of MIH, IT injection of WIN 55,212-2 combined with EA induced a significant increase in MWT and TWL accompanied with a significant decrease in p-ERK1/2 and a significant increase in CB1 protein level compared with EA alone, while SR141716 induced the opposite results. The present study suggests that EA alleviates hyperalgesia induced by IT injection of morphine partially through the inhibition of ERK1/2 activation. Activation of the CB1 receptor enhances the antinociceptive effect of EA in rats with MIH partly through the regulation of the spinal CB1-ERK1/2 signaling pathway.

Impaired Ethanol-Induced Sensitization and Decreased Cannabinoid Receptor-1 in a Model of Posttraumatic Stress Disorder.

Background and PurposeImpaired striatal neuroplasticity may underlie increased alcoholism documented in those with posttraumatic stress disorder (PTSD). Cannabinoid receptor-1 (CB1) is sensitive to the effects of ethanol (EtOH) and traumatic stress, and is a critical regulator of striatal plasticity. To investigate CB1 involvement in the PTSD-alcohol interaction, this study measured the effects of traumatic stress using a model of PTSD, mouse single-prolonged stress (mSPS), on EtOH-induced locomotor sensitization and striatal CB1 levels.MethodsMice were exposed to mSPS, which includes: 2-h restraint, 10-min group forced swim, 15-min exposure to rat bedding odor, and diethyl ether exposure until unconsciousness or control conditions. Seven days following mSPS exposure, the locomotor sensitizing effects of EtOH were assessed. CB1, post-synaptic density-95 (PSD95), and dopamine-2 receptor (D2) protein levels were then quantified in the dorsal striatum using standard immunoblotting techniques.ResultsMice exposed to mSPS-EtOH demonstrated impaired EtOH-induced locomotor sensitization compared to Control-EtOH mice, which was accompanied by reduced striatal CB1 levels. EtOH increased striatal PSD95 in control and mSPS-exposed mice. Additionally, mSPS-Saline exposure increased striatal PSD95 and decreased D2 protein expression, with mSPS-EtOH exposure alleviating these changes.ConclusionsThese data indicate that the mSPS model of PTSD blunts the behavioral sensitizing effects of EtOH, a response that suggests impaired striatal neuroplasticity. Additionally, this study demonstrates that mice exposed to mSPS and repeated EtOH exposure decreases CB1 in the striatum, providing a mechanism of interest for understanding the effects of EtOH following severe, multimodal stress exposure.

191 SEARCHING FOR THE IN VIVO TRANSCRIPTOME BLUEPRINT OF COMPETENT BOVINE OOCYTES

Gene expression in early stage embryos relies mostly on post-transcriptional control of maternal transcripts accumulated during oocyte maturation. However, while the building process to obtain a competent oocyte is now better understood, it is still not clear what transcriptome blueprint composes a competent oocyte. The aim of the study was to compare the mRNA expression pattern between oocytes collected from fertile heifers and repeat breeders by using RNAseq. Oocytes were collected by ovum pickup from 3 heifers that were 11–15 months of age and became pregnant at the following oestrus and from 4 adult cows with an age of 4 to 7 years, classified as repeat breeders after they failed to become pregnant for a minimum of 3 consecutive AI. To obtain oocytes from follicles with the same degree of development, at time 0 all follicles visible through transrectal ultrasound examination were removed by transvaginal aspiration. Five days later oocytes were collected by ovum pick up from the newly formed follicles with diameters >5 mm. Oocytes from each animal were pooled and sequenced as a single sample. Total RNA was extracted by RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Amplified cDNA, was prepared starting from total RNA using the Ovation RNA-Seq System V2 (Nugen Technologies, San Carlos, CA, USA). After library preparation with TruSeq DNA Sample Prep kit (Illumina, Madison, WI, USA), sequencing was performed on an Illumina HiSEqn 2000. Galaxy and Chipster open web-based platforms were used to analyse the data. We identified 49 differentially expressed genes. Heifers’ oocytes mRNA pattern indicated greater potential to sustain cell division. In particular, oocytes expressed more Keratin 14 (a gene involved in cell proliferation) and kinesin family member 20B (a protein involved in cytokinesis). More competent oocytes also have a greater ability to repair single-strand breaks due to the high levels of endo/exonuclease (5′-3′), endonuclease G-like. This may reflect greater capacity to neutralise DNA damage and, therefore, greater ability to preserve and transmit high-quality DNA. Repeat breeders portray a different landscape; their greater expression of Jun oncogene, Heat shock protein 1, Stimulated by retinoic acid gene6, arylhydrocarbon receptor nuclear translocator, fibromodulin, and aryl-hydrocarbon receptor repressor suggest that these oocytes have been subjected to environmental stress during oocyte maturation. Their greater expressions of inhibin α, stearoyl-CoA desaturase, junctional adhesion molecule 2 have been previously shown to correlate with a reduced oocyte developmental potential. Furthermore, the Cannabinoid receptor protein 1 expression suggests a compromised ion function that can lead to a failed activation of the development program. Finally, the greater expression of Ubiquilin3 and Heat shock protein 1 led to high protein and mRNA degradation, respectively, suggesting that these oocytes are deprived of essential components to sustain embryo growth. In conclusion our data provide the first detailed snapshot of the mRNA pattern defining the differences between a competent oocyte and an incompetent oocyte in vivo. Study supported by PRIN 2008, 2009 and EU-Quantomics.

O11. Evaluation of the endocannabinoid system in preeclampsia

Introduction The endocannabinoid system plays a key role in female reproduction, including implantation, decidualization and placentation. A growing number of studies indicate that placental and peripheral blood anandamide levels correlate closely with both spontaneous miscarriage and ectopic pregnancy. Anandamide has also been implicated in blood pressure regulation. Objectives In this study, we aimed to analyze placental expression and localization of cannabinoid receptor 1 (CB1), CB2 and fatty acid amid hydrolase (FAAH), as well as circulating anandamide levels in normal pregnancy and preeclampsia. Methods We determined CB1, CB2 and FAAH expressions by Western blotting and immunohistochemistry in placental samples collected directly after Cesarean section in 18 preeclamptic patients and 18 normotensive, healthy pregnant women. Serum anandamide concentrations were measured by high performance liquid chromatography–mass spectrometry (HPLC–MS) technique in 43 preeclamptic patients and 71 healthy pregnant women. Serum total soluble fms-like tyrosine kinase-1 (sFlt-1) and biologically active placental growth factor (PlGF) levels were assessed by electrochemiluminescence immunoassay. Results CB1 expression semi-quantified by Western blotting was significantly higher in preeclamptic placenta, and these findings were confirmed by immunohistochemistry. CB1 immunoreactivity was markedly stronger in syncytiotrophoblasts, the mesenchymal core, decidua, villous capillary endothelial and smooth muscle cells, as well as in the amnion in preeclamptic samples compared to normal pregnancies. However, we did not find significant differences between preeclamptic and normal placenta in terms of CB2 and FAAH expressions and immunoreactivity. Serum levels of anandamide were significantly lower in preeclamptic patients than in healthy pregnant women. Preeclamptic patients had significantly higher sFlt-1 levels and significantly lower PlGF concentrations as compared to healthy pregnant women. Serum anandamide concentrations did not correlate with serum levels of sFlt-1 and PlGF in our healthy pregnant and preeclamptic groups. Conclusion We observed markedly higher expression of CB1 protein in preeclamptic placental tissue. Increased CB1 expression might cause abnormal decidualization and impair trophoblast invasion, thus being involved in the pathogenesis of preeclampsia. Nevertheless, we did not find significant differences between preeclamptic and normal placental tissue regarding CB2 and FAAH expressions. Furthermore, serum anandamide concentrations were decreased in women with preeclampsia. While the detailed pathogenesis of preeclampsia is still unclear, the endocannabinoid system could play a role in the development of the disease.

Role of cannabinoid receptor CB2 in HER2 pro-oncogenic signaling in breast cancer.

Pharmacological activation of cannabinoid receptors elicits antitumoral responses in different cancer models. However, the biological role of these receptors in tumor physio-pathology is still unknown. We analyzed CB2 cannabinoid receptor protein expression in two series of 166 and 483 breast tumor samples operated in the University Hospitals of Kiel, Tübingen, and Freiburg between 1997 and 2010 and CB2 mRNA expression in previously published DNA microarray datasets. The role of CB2 in oncogenesis was studied by generating a mouse line that expresses the human V-Erb-B2 Avian Erythroblastic Leukemia Viral Oncogene Homolog 2 (HER2) rat ortholog (neu) and lacks CB2 and by a variety of biochemical and cell biology approaches in human breast cancer cells in culture and in vivo, upon modulation of CB2 expression by si/shRNAs and overexpression plasmids. CB2-HER2 molecular interaction was studied by colocalization, coimmunoprecipitation, and proximity ligation assays. Statistical tests were two-sided. We show an association between elevated CB2 expression in HER2+ breast tumors and poor patient prognosis (decreased overall survival, hazard ratio [HR] = 0.29, 95% confidence interval [CI] = 0.09 to 0.71, P = .009) and higher probability to suffer local recurrence (HR = 0.09, 95% CI = 0.049 to 0.54, P = .003) and to develop distant metastases (HR = 0.33, 95% CI = 0.13 to 0.75, P = .009). We also demonstrate that genetic inactivation of CB2 impairs tumor generation and progression in MMTV-neu mice. Moreover, we show that HER2 upregulates CB2 expression by activating the transcription factor ELK1 via the ERK cascade and that an increased CB2 expression activates the HER2 pro-oncogenic signaling at the level of the tyrosine kinase c-SRC. Finally, we show HER2 and CB2 form heteromers in cancer cells. Our findings reveal an unprecedented role of CB2 as a pivotal regulator of HER2 pro-oncogenic signaling in breast cancer, and they suggest that CB2 may be a biomarker with prognostic value in these tumors.

Elevated cannabinoid receptor 1 and G protein-coupled receptor 55 expression in proximal tubule cells and whole kidney exposed to diabetic conditions.

Hyperglycaemia increases the risk of developing diabetic nephropathy, with primary targets in the glomerulus and proximal tubule. Importantly, glomerular damage in the kidney leads to elevated albumin levels in the filtrate, which contributes to tubular structural modifications that lead to dysfunction. Diabetes alters the endocannabinoid system in a number of target organs, with previous research characterizing tissue-specific changes in the expression of the cannabinoid receptor 1 (CB1 ) and G protein-coupled receptor 55 (GPR55), a putative cannabinoid receptor, in diabetes. Although these receptors have a functional role in the cannabinoid system in the kidney, there has been little investigation into changes in the expression of CB1 and GPR55 in the proximal tubule under diabetic conditions. In this study, CB1 and GPR55 messenger RNA and protein levels were quantified in cultured human kidney cells and then treated with either elevated glucose, elevated albumin, or a combination of glucose and albumin for 4, 6, 18, or 24h. In addition, CB1 and GPR55 protein expression was characterized in whole-kidney lysate from streptozotocin-induced diabetic Sprague-Dawley rats. In vitro exposure to elevated glucose and albumin increased CB1 and GPR55 messenger RNA and protein expression in proximal tubule cells in a time-dependant manner. In whole kidney of streptozotocin-induced diabetic rats, CB1 protein was upregulated, whereas GPR55 protein concentration was not altered. Thus, expression of CB1 and GPR55 in proximal tubules is altered in response to elevated levels of glucose and albumin. Further investigations should determine if these receptors are effective physiological targets for the treatment and prevention of diabetic nephropathy.

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Objectives The endocannabinoid system plays a key role in female reproduction, including implantation, decidualization and placentation. In the present study, we aimed to analyze cannabinoid receptor 1 (CB1), CB2 and fatty acid amid hydrolase (FAAH) expressions and localization in normal and preeclamptic placenta, in order to determine whether aberrant endocannabinoid activity is related to preeclampsia. Methods Eighteen preeclamptic patients and 18 normotensive, healthy pregnant women with uncomplicated pregnancies were involved in our case-control study. We determined CB1, CB2 and FAAH expressions by Western immunoblotting and immunohistochemistry in placental samples collected directly after Cesarean section. Results CB1 expression measured by Western immunoblotting was significantly higher in preeclamptic placenta, and these findings were confirmed by immunohistochemistry. CB1 immunoreactivity was markedly stronger in syncytiotrophoblasts, the mesenchymal core, decidua, villous capillary endothelial and smooth muscle cells, as well as in the amnion in preeclamptic samples compared to normal pregnancies. However, we did not find significant differences between preeclamptic and normal placenta in terms of CB2 and FAAH expressions and immunoreactivity. Conclusions We observed markedly higher expression of CB1 protein in preeclamptic placental tissue. Increased CB1 expression might cause abnormal decidualization and impair trophoblast invasion, thus being involved in the pathogenesis of preeclampsia. As CB1 activation can induce endothelial dysfunction and enhance vascular inflammation, the strong CB1 immunoreaction in vascular endothelial and smooth muscle cells suggests that CB1 may contribute to the development of atherosis in the placental villi shown earlier in preeclampsia. While the detailed pathogenesis of preeclampsia is still unclear, the endocannabinoid system could play a role in the development of the disease. Disclosures A. Molvarec: None. G. Fugedi: None. M. Molnar: None. J. Schonleber: None. I. Kovalszky: None. J. Rigo: None.

Increased placental expression of cannabinoid receptor 1 in preeclampsia: an observational study.

BackgroundThe endocannabinoid system plays a key role in female reproduction, including implantation, decidualization and placentation. In the present study, we aimed to analyze cannabinoid receptor 1 (CB1), CB2 and fatty acid amid hydrolase (FAAH) expressions and localization in normal and preeclamptic placenta, in order to determine whether placental endocannabinoid expression pattern differs between normal pregnancy and preeclampsia.MethodsEighteen preeclamptic patients and 18 normotensive, healthy pregnant women with uncomplicated pregnancies were involved in our case–control study. We determined CB1, CB2 and FAAH expressions by Western blotting and immunohistochemistry in placental samples collected directly after Cesarean section.ResultsCB1 expression semi-quantified by Western blotting was significantly higher in preeclamptic placenta, and these findings were confirmed by immunohistochemistry. CB1 immunoreactivity was markedly stronger in syncytiotrophoblasts, the mesenchymal core, decidua, villous capillary endothelial and smooth muscle cells, as well as in the amnion in preeclamptic samples compared to normal pregnancies. However, we did not find significant differences between preeclamptic and normal placenta in terms of CB2 and FAAH expressions and immunoreactivity.ConclusionsWe observed markedly higher expression of CB1 protein in preeclamptic placental tissue. Increased CB1 expression might cause abnormal decidualization and impair trophoblast invasion, thus being involved in the pathogenesis of preeclampsia. Nevertheless, we did not find significant differences between preeclamptic and normal placental tissue regarding CB2 and FAAH expressions. While the detailed pathogenesis of preeclampsia is still unclear, the endocannabinoid system could play a role in the development of the disease.

Expression and Functional Relevance of Cannabinoid Receptor 1 in Hodgkin Lymphoma

BackgroundCannabinoid receptor 1 (CB1) is expressed in certain types of malignancies. An analysis of CB1 expression and function in Hodgkin lymphoma (HL), one of the most frequent lymphomas, was not performed to date.Design and MethodsWe examined the distribution of CB1 protein in primary cases of HL. Using lymphoma derived cell lines, the role of CB1 signaling on cell survival was investigated.ResultsA predominant expression of CB1 was found in Hodgkin-Reed-Sternberg cells in a vast majority of classical HL cases. The HL cell lines L428, L540 and KM-H2 showed strong CB1-abundance and displayed a dose-dependent decline of viability under CB1 inhibition with AM251. Further, application of AM251 led to decrease of constitutively active NFκB/p65, a crucial survival factor of HRS-cells, and was followed by elevation of apoptotic markers in HL cells.ConclusionsThe present study identifies CB1 as a feature of HL, which might serve as a potential selective target in the treatment of Hodgkin lymphoma.

Differential expression of intracellular and extracellular CB(2) cannabinoid receptor protein by human peripheral blood leukocytes.

mRNA encoding for the CB(2) cannabinoid receptor is expressed by many subsets of human peripheral blood leukocytes (PBL), but little is known about the resulting protein expression and function. Employing clones from the A549 and 293T cell lines that were constructed to express both full-length human CB(2) and GFP, we developed a flow cytometry assay for characterizing CB(2) protein expression. A monoclonal antibody directed against human CB(2) selectively stained the surface of transduced but not parental cell lines. When cells were fixed and permeabilized, imaging flow cytometry identified large stores of intracellular protein. Total cellular staining for CB(2) corresponded closely with the level of GFP expression. When exposed to Δ(9)-tetrahydrocannabinol, CB(2)-expressing cells internalized cell surface CB(2) receptors in a time- and dose-dependent manner. Applying these approaches to human PBL, CB(2) protein was identified on the surface of human B cells but not on T cells or monocytes. In contrast, when PBL were fixed and permeabilized, intracellular CB(2) expression was readily detected in all three subsets by both conventional and imaging flow cytometry. Similar to the protein expression pattern observed in fixed and permeabilized PBL, purified B cells, T cells, and monocytes expressed relatively equal levels of CB(2) mRNA by quantitative real-time RT-PCR. Our findings confirm that human PBL express CB(2) protein but that its distribution is predominantly intracellular with only B cells expressing CB(2) protein at the extracellular membrane. The differential role of intracellular and extracellular CB(2) receptors in mediating ligand signaling and immune function remains to be determined.

A Novel Monoclonal Antibody Against Cannabinoid Receptor 1

The cannabinoid receptor 1 (CBR1) is being widely investigated because of its specific structure and functions compared with other cannabinoid receptors. In this study, we immunized BALB/c mice with synthesized human CBR1 polypeptide and obtained a novel monoclonal antibody (MAb) against human CBR1. Analysis through enzyme-linked immunosorbent assay (ELISA), spot-ELISA, Western blot, and immunohistochemistry revealed that the MAb was specifically against recombinant human CBR1 protein, and its subtype and affinity constant (Kaff) were IgG2b/k and 7.85 × 10(8) M/L, respectively. Using this MAb we found that CBR1 is expressed on HL-7702 cells and lipid tissue, raising the possibility that the CBR1 may take a role in glucose and lipid metabolism. Thus, this antibody might facilitate studies for pathophysiology of diseases associated with glucose and lipid metabolism abnormality.

Signal peptide and denaturing temperature are critical factors for efficient mammalian expression and immunoblotting of cannabinoid receptors

Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e., ≥95°C), forming a large molecular weight band when analyzed by immuno-blotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immuno-blot analysis. These findings provide very useful information for efficient mammalian expression and immuno-blotting of membrane receptors.

The localization and physiological effects of cannabinoid receptor 1 in the brain stem auditory system of the chick

Fast, temporally-precise, and consistent synaptic transmission is required to encode features of acoustic stimuli. Neurons of nucleus magnocellularis (NM) in the auditory brain stem of the chick possess numerous adaptations to optimize the coding of temporal information. One potential problem for the system is the depression of synaptic transmission during a prolonged stimulus. The present study tested the hypothesis that cannabinoid receptor 1 (CB1) signaling may limit synaptic depression at the auditory nerve-NM synapse. In situ hybridization was used to confirm that CB1 mRNA is expressed in the cochlear ganglion; immunohistochemistry was used to confirm the presence of CB1 protein in NM. These findings are consistent with the common presynaptic locus of CB1 in the brain. Rate-dependent synaptic depression was then examined in a brain slice preparation before and after administration of WIN 55,212-2 (WIN), a potent CB1 agonist. WIN decreased the amplitude of excitatory postsynaptic currents (EPSCs) and also reduced depression across a train of stimuli. The effect was most obvious late in the pulse train and during high rates of stimulation. This CB1-mediated influence could allow for lower, but more consistent activation of NM neurons, which could be of importance for optimizing the coding of prolonged, temporally-locked acoustic stimuli.

Molecular reorganization of endocannabinoid signalling in Alzheimer’s disease

Retrograde messengers adjust the precise timing of neurotransmitter release from the presynapse, thus modulating synaptic efficacy and neuronal activity. 2-Arachidonoyl glycerol, an endocannabinoid, is one such messenger produced in the postsynapse that inhibits neurotransmitter release upon activating presynaptic CB(1) cannabinoid receptors. Cognitive decline in Alzheimer's disease is due to synaptic failure in hippocampal neuronal networks. We hypothesized that errant retrograde 2-arachidonoyl glycerol signalling impairs synaptic neurotransmission in Alzheimer's disease. Comparative protein profiling and quantitative morphometry showed that overall CB(1) cannabinoid receptor protein levels in the hippocampi of patients with Alzheimer's disease remain unchanged relative to age-matched controls, and CB(1) cannabinoid receptor-positive presynapses engulf amyloid-β-containing senile plaques. Hippocampal protein concentrations for the sn-1-diacylglycerol lipase α and β isoforms, synthesizing 2-arachidonoyl glycerol, significantly increased in definite Alzheimer's (Braak stage VI), with ectopic sn-1-diacylglycerol lipase β expression found in microglia accumulating near senile plaques and apposing CB(1) cannabinoid receptor-positive presynapses. We found that microglia, expressing two 2-arachidonoyl glycerol-degrading enzymes, serine hydrolase α/β-hydrolase domain-containing 6 and monoacylglycerol lipase, begin to surround senile plaques in probable Alzheimer's disease (Braak stage III). However, Alzheimer's pathology differentially impacts serine hydrolase α/β-hydrolase domain-containing 6 and monoacylglycerol lipase in hippocampal neurons: serine hydrolase α/β-hydrolase domain-containing 6 expression ceases in neurofibrillary tangle-bearing pyramidal cells. In contrast, pyramidal cells containing hyperphosphorylated tau retain monoacylglycerol lipase expression, although at levels significantly lower than in neurons lacking neurofibrillary pathology. Here, monoacylglycerol lipase accumulates in CB(1) cannabinoid receptor-positive presynapses. Subcellular fractionation revealed impaired monoacylglycerol lipase recruitment to biological membranes in post-mortem Alzheimer's tissues, suggesting that disease progression slows the termination of 2-arachidonoyl glycerol signalling. We have experimentally confirmed that altered 2-arachidonoyl glycerol signalling could contribute to synapse silencing in Alzheimer's disease by demonstrating significantly prolonged depolarization-induced suppression of inhibition when superfusing mouse hippocampi with amyloid-β. We propose that the temporal dynamics and cellular specificity of molecular rearrangements impairing 2-arachidonoyl glycerol availability and actions may differ from those of anandamide. Thus, enhanced endocannabinoid signalling, particularly around senile plaques, can exacerbate synaptic failure in Alzheimer's disease.