Effects of Cannabinoids on Tension Induced by Acetylcholine and Choline in Slow Skeletal Muscle Fibers of the Frog

We investigated the effects of cannabinoids on acetylcholine (ACh) or choline contractures in slow skeletal muscle fibers from Rana pipiens. Bundles of cruralis muscle fibers were incubated with the cannabinoid receptor 1 (CB₁) agonist, arachidonylcyclopropylamide (ACPA), which diminished the maximum isometric tension by 10 % and the total tension by 5 % of the ACh contracture, and 40 and 22 % of the choline contracture, respectively. Preincubation with the CB₁ antagonist, AM281, or with pertussis toxin (PTX) completely blocked the effect of ACPA on the ACh contracture. On the other hand, the decrease in choline contracture by ACPA was only partially blocked by AM281 (~16 % decrease), PTX (20 %), or by dantrolene (~46 %). Our results show that ACPA modulates ACh and choline contractures, and suggest that this effect involves the participation of CB₁, the ACh receptor, and ?RyR in ACh contractures. For choline contractures, ACPA may also be acting through cannabinoid receptor-independent mechanisms.     

Down-Regulation of Adiponectin Receptors in Osteoarthritic Chondrocytes

The objective of this study was to investigate the expression of adiponectin receptors (AdipoR1, R2, and T-cadherin) in both normal subjects and patients with knee osteoarthritis (OA). We used immunofluorescence to assess expression of adiponectin receptors in the chondrocytes of normal subjects (n = 3) and OA patients (n = 3). We also studied mRNA expression of adiponectin receptors in both groups by real-time polymerase chain reaction (real-time PCR). Finally, we utilized Western blotting to confirm the presence of adiponectin receptors. As compared with osteoarthritic chondrocytes, normal chondrocytes showed stronger immunoreactivity for AdipoR1, AdipoR2, and T-cadherin. The expression levels of both AdipoR1 and AdipoR2 mRNA were significantly lower in the osteoarthritic chondrocytes compared with those in the normal chondrocytes, 19 ± 2 and 36 ± 3 % of normal chondrocytes, respectively (P < 0.001). T-cadherin mRNA expression levels of the osteoarthritic chondrocytes were also lower than those in the normal chondrocytes, but not statistical significant (P = 0.072). The expression levels of AdipoR1 and AdipoR2 protein were significantly higher in the normal chondrocytes compared with those in the osteoarthritic chondrocytes (P < 0.001, P < 0.01, respectively). T-cadherin protein expression level of the normal chondrocytes was also higher than those in the osteoarthritic chondrocytes, but the difference is not statistical significant (P = 0.114). Expression of adiponectin receptors protein in normal and osteoarthritic chondrocytes is consistent with its mRNA expression levels. In conclusion, we report for the first time down-regulation of adiponectin receptors (AdipoR1, R2, and T-cadherin) in osteoarthritic chondrocytes. Decreased adiponectin receptors in OA may reduce the tissue sensitivity to adiponectin, thus lost the protection from adiponectin in the progression of OA.     

CB2 Cannabinoid Receptors Contribute to Bacterial Invasion and Mortality in Polymicrobial Sepsis

Background

Sepsis is a major healthcare problem and current estimates suggest that the incidence of sepsis is approximately 750,000 annually. Sepsis is caused by an inability of the immune system to eliminate invading pathogens. It was recently proposed that endogenous mediators produced during sepsis can contribute to the immune dysfunction that is observed in sepsis. Endocannabinoids that are produced excessively in sepsis are potential factors leading to immune dysfunction, because they suppress immune cell function by binding to G-protein-coupled CB₂ receptors on immune cells. Here we examined the role of CB₂ receptors in regulating the host''s response to sepsis.

Methods and Findings

The role of CB₂ receptors was studied by subjecting CB₂ receptor wild-type and knockout mice to bacterial sepsis induced by cecal ligation and puncture. We report that CB₂ receptor inactivation by knockout decreases sepsis-induced mortality, and bacterial translocation into the bloodstream of septic animals. Furthermore, CB₂ receptor inactivation decreases kidney and muscle injury, suppresses splenic nuclear factor (NF)-κB activation, and diminishes the production of IL-10, IL-6 and MIP-2. Finally, CB₂ receptor deficiency prevents apoptosis in lymphoid organs and augments the number of CD11b⁺ and CD19⁺ cells during CLP.

Conclusions

Taken together, our results establish for the first time that CB₂ receptors are important contributors to septic immune dysfunction and mortality, indicating that CB₂ receptors may be therapeutically targeted for the benefit of patients suffering from sepsis.     

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

Cannabinoid CB₁ receptors peripherally modulate energy metabolism. Here, we investigated the role of CB₁ receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of α-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB₁ receptor antagonist AM251 (3 mg kg^-1, 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle—regulated by both diet and CB₁ receptor activity—through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB₁ receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB₁ receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB ₁ ^-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C₂C₁₂ myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB₁ receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.     

iPLA2β Knockout Mouse,a Genetic Model for Progressive Human Motor Disorders,Develops Age-Related Neuropathology

Calcium-independent phospholipase A₂ group VIa (iPLA₂β) preferentially releases docosahexaenoic acid (DHA) from the sn-2 position of phospholipids. Mutations of its gene, PLA2G6, are found in patients with several progressive motor disorders, including Parkinson disease. At 4 months, PLA2G6 knockout mice (iPLA₂β^?/?) show minimal neuropathology but altered brain DHA metabolism. By 1 year, they develop motor disturbances, cerebellar neuronal loss, and striatal α-synuclein accumulation. We hypothesized that older iPLA₂β^?/? mice also would exhibit inflammatory and other neuropathological changes. Real-time polymerase chain reaction and Western blotting were performed on whole brain homogenate from 15 to 20-month old male iPLA₂β^?/? or wild-type (WT) mice. These older iPLA₂β^?/? mice compared with WT showed molecular evidence of microglial (CD-11b, iNOS) and astrocytic (glial fibrillary acidic protein) activation, disturbed expression of enzymes involved in arachidonic acid metabolism, loss of neuroprotective brain derived neurotrophic factor, and accumulation of cytokine TNF-α messenger ribonucleic acid, consistent with neuroinflammatory pathology. There was no evidence of synaptic loss, of reduced expression of dopamine active reuptake transporter, or of accumulation of the Parkinson disease markers Parkin or Pink1. iPLA₂γ expression was unchanged. iPLA₂β deficient mice show evidence of neuroinflammation and associated neuropathology with motor dysfunction in later life. These pathological biomarkers could be used to assess efficacy of dietary intervention, antioxidants or other therapies on disease progression in this mouse model of progressive human motor diseases associated with a PLA2G6 mutation.     

Probiotic Properties of Lactobacillus crispatus 2,029: Homeostatic Interaction with Cervicovaginal Epithelial Cells and Antagonistic Activity to Genitourinary Pathogens

Lactobacillus crispatus 2029 isolated upon investigation of vaginal lactobacilli of healthy women of reproductive age was selected as a probiotic candidate. The aim of the present study was elucidation of the role of L. crispatus 2029 in resistance of the female reproductive tract to genitourinary pathogens using cervicovaginal epithelial model. Lactobacillus crispatus 2029 has surface layers (S-layers), which completely surround cells as the outermost component of their envelope. S-layers are responsible for the adhesion of lactobacilli on the surface of cervicovaginal epithelial cells. Study of interactions between L. crispatus 2029 and a type IV collagen, a major molecular component of epithelial cell extracellular matrix, showed that 125I-labeled type IV collagen binds to lactobacilli with high affinity (Kd = (8.0 ± 0.7) × 10^?10 M). Lactobacillus crispatus 2029 consistently colonized epithelial cells. There were no toxicity, epithelial damage and apoptosis after 24 h of colonization. Electronic microscope images demonstrated intimate association between L. crispatus 2029 and epithelial cells. Upon binding to epithelial cells, lactobacilli were recognized by toll-like 2/6 receptors. Lactobacillus crispatus induced NF-κB activation in epithelial cells and did not induce expression of innate immunity mediators IL-8, IL-1β, IL-1α and TNF-α. Lactobacillus crispatus 2029 inhibited IL-8 production in epithelial cells induced by MALP-2 and increased production of anti-inflammatory cytokine IL-6, maintaining the homeostasis of female reproductive tract. Lactobacillus crispatus 2029 produced H₂O₂ and provided wide spectrum of antagonistic activity increasing colonization resistance to urinary tract infections by bacterial vaginosis and vulvovaginal candidiasis associated agents.     

The Expression Level of CB1 and CB2 Receptors Determines Their Efficacy at Inducing Apoptosis in Astrocytomas

Background

Cannabinoids represent unique compounds for treating tumors, including astrocytomas. Whether CB₁ and CB₂ receptors mediate this therapeutic effect is unclear.

Principal Findings

We generated astrocytoma subclones that express set levels of CB₁ and CB₂, and found that cannabinoids induce apoptosis only in cells expressing low levels of receptors that couple to ERK1/2. In contrast, cannabinoids do not induce apoptosis in cells expressing high levels of receptors because these now also couple to the prosurvival signal AKT. Remarkably, cannabinoids applied at high concentration induce apoptosis in all subclones independently of CB₁, CB₂ and AKT, but still through a mechanism involving ERK1/2.

Significance

The high expression level of CB₁ and CB₂ receptors commonly found in malignant astrocytomas precludes the use of cannabinoids as therapeutics, unless AKT is concomitantly inhibited, or cannabinoids are applied at concentrations that bypass CB₁ and CB₂ receptors, yet still activate ERK1/2.     

Activation of GABAB Receptors Ameliorates Cognitive Impairment via Restoring the Balance of HCN1/HCN2 Surface Expression in the Hippocampal CA1 Area in Rats With Chronic Cerebral Hypoperfusion

Hyperpolarization-activated cyclic-nucleotide-gated cation nonselective (HCN) channels are involved in the pathology of nervous system diseases. HCN channels and γ-aminobutyric acid (GABA) receptors can mutually co-regulate the function of neurons in many brain areas. However, little is known about the co-regulation of HCN channels and GABA receptors in the chronic ischemic rats with possible features of vascular dementia. Protein kinase A (PKA) and TPR containing Rab8b interacting protein (TRIP8b) can modulate GABA_B receptors cell surface stability and HCN channel trafficking, respectively, and adaptor-associated kinase 1 (AAK1) inhibits the function of the major TRIP8b-interacting protein adaptor protein 2 (AP2) via phosphorylating the AP2 μ2 subunit. Until now, the role of these regulatory factors in chronic cerebral hypoperfusion is unclear. In the present study, we evaluated whether and how HCN channels and GABA_B receptors were pathologically altered and investigated neuroprotective effects of GABA_B receptors activation and cross-talk networks between GABA_B receptors and HCN channels in the hippocampal CA1 area in chronic cerebral hypoperfusion rat model. We found that cerebral hypoperfusion for 5 weeks by permanent occlusion of bilateral common carotid arteries (two-vessel occlusion, 2VO) induced marked spatial and nonspatial learning and memory deficits, significant neuronal loss and decrease in dendritic spine density, impairment of long-term potentiation (LTP) at the Schaffer collateral-CA1 synapses, and reduction of surface expression of GABA_B R1, GABA_B R2, and HCN1, but increase in HCN2 surface expression. Meanwhile, the protein expression of TRIP8b (1a-4), TRIP8b (1b-2), and AAK1 was significantly decreased. Baclofen, a GABA_B receptor agonist, markedly improved the memory impairment and alleviated neuronal damage. Besides, baclofen attenuated the decrease of surface expression of GABA_B R1, GABA_B R2, and HCN1, but downregulated HCN2 surface expression. Furthermore, baclofen could restore expression of AAK1 protein and significantly increase p-PKA, TRIP8b (1a-4), TRIP8b (1b-2), and p-AP2 μ2 expression. Those findings suggested that, under chronic cerebral hypoperfusion, activation of PKA could attenuate baclofen-induced decrease in surface expression of GABA_B R1 and GABA_B R2, and activation of GABA_B receptors not only increased the expression of TRIP8b (1a-4) and TRIP8b (1b-2) but also regulated the function of TRIP8b via AAK1 and p-AP2 μ2, which restored the balance of HCN1/HCN2 surface expression in rat hippocampal CA1 area, and thus ameliorated cognitive impairment.     

Paracrine Transactivation of the CB1 Cannabinoid Receptor by AT1 Angiotensin and Other Gq/11 Protein-coupled Receptors

Intracellular signaling systems of G protein-coupled receptors are well established, but their role in paracrine regulation of adjacent cells is generally considered as a tissue-specific mechanism. We have shown previously that AT₁ receptor (AT₁R) stimulation leads to diacylglycerol lipase-mediated transactivation of co-expressed CB₁Rs in Chinese hamster ovary cells. In the present study we detected a paracrine effect of the endocannabinoid release from Chinese hamster ovary, COS7, and HEK293 cells during the stimulation of AT₁ angiotensin receptors by determining CB₁ cannabinoid receptor activity with bioluminescence resonance energy transfer-based sensors of G protein activation expressed in separate cells. The angiotensin II-induced, paracrine activation of CB₁ receptors was visualized by detecting translocation of green fluorescent protein-tagged β-arrestin2. Mass spectrometry analyses have demonstrated angiotensin II-induced stimulation of 2-arachidonoylglycerol production, whereas no increase of anandamide levels was observed. Stimulation of G_q/11-coupled M₁, M₃, M₅ muscarinic, V₁ vasopressin, α_1a adrenergic, B₂ bradykinin receptors, but not G_i/o-coupled M₂ and M₄ muscarinic receptors, also led to paracrine transactivation of CB₁ receptors. These data suggest that, in addition to their retrograde neurotransmitter role, endocannabinoids have much broader paracrine mediator functions during activation of G_q/11-coupled receptors.Hormones, neurotransmitters, and other chemical mediators acting on G protein-coupled receptors (GPCRs)² exert their effects on the target cells by stimulating G protein-dependent and independent intracellular signaling pathways (1–4). Activation of G_q/11 protein-coupled receptors causes phospholipase C activation, which produces inositol-trisphosphate and diacylglycerol from phosphatidylinositol (4,5)-bisphosphate, leading to Ca²⁺-signal generation and protein kinase C activation. However, the concerted response of tissues to chemical mediators frequently also involves the activation of cells adjacent to the target cells, due to the release of paracrine mediators. A well known example is NO, which can be released from activated endothelial cells to cause relaxation of adjacent vascular smooth muscle cells. Lipid mediators can also act as intercellular messengers. For example, endocannabinoids released from postsynaptic neurons after depolarization act as retrograde transmitters by binding to and stimulating presynaptic cannabinoid receptors, which leads to inhibition of γ-aminobutyric acid release (an event termed depolarization-induced suppression of inhibition, DSI) (5–7).Cannabinoid receptors were first identified based on their ability to selectively recognize marijuana analogs. To date, two cannabinoid receptors have been identified by molecular cloning, CB₁ and CB₂ receptors (CB₁R and CB₂R, respectively) (5, 8, 9), although additional GPCRs have also been proposed to function as cannabinoid receptors (10, 11). Cannabinoid receptors also recognize certain lipids present in animal tissues termed endocannabinoids, such as arachidonylethanolamide (anandamide), 2-arachidonoylglycerol (2-AG), and 2-arachidonoylglyceryl ether (noladin ether) (7, 12–16). In adult and fetal neural tissues, the two major endocannabinoids, anandamide and 2-AG, are produced on demand, usually after depolarization of postsynaptic cells or following stimulation of G_q-coupled metabotropic glutamate or muscarinic acetylcholine receptors (7, 12, 17–20). Enzymes responsible for 2-AG production and metabolism in tissues are localized to well defined structures at synapses, near the axon terminals of CB₁R-expressing cells (5, 7). In contrast, in peripheral tissues baseline levels of endocannabinoid production usually manifest as “endocannabinoid tone,” with poorly understood localization of the various components of the endocannabinoid system. 2-AG levels in brain homogenates and in many peripheral tissues are near its K_d for the CB₁R (19), suggesting that function of endocannabinoids may not be limited to localized synaptic signaling.There is mounting evidence that endocannabinoids play important roles in peripheral cardiovascular, inflammatory, intestinal, and metabolic regulation (21–24). 2-AG is produced by diacylglycerol-lipase (DAGL) after cleavage of the fatty-acid in the sn-1 position of diacylglycerol (DAG) (19, 25). Phospholipase C activation by G_q/11 protein-coupled receptors produces DAG, which can serve as a substrate for DAGL. Plasma membrane phosphoinositides are enriched in arachidonic acid in the sn-2 position (26), and DAGL is expressed ubiquitously (27), which suggests that phospholipase C-mediated cleavage of polyphosphoinositides may routinely lead to the formation of 2-AG. In accordance with this hypothesis, we have recently shown that angiotensin II- (Ang II)-mediated activation of the G_q/11-coupled AT₁ angiotensin receptor (AT₁R) leads to DAGL-dependent activation of CB₁Rs expressed in Chinese hamster ovary (CHO) cells (28).Here our aim has been to examine the possibility that 2-AG serves as a common paracrine signal generated via activation of G_q/11 protein-coupled, Ca²⁺-mobilizing receptors. Accordingly, we co-expressed CB₁Rs and BRET-based sensors of G protein activation in CHO cells, and used these cells to detect endocannabinoid release from adjacent cells that express AT₁R or other Ca²⁺-mobilizing GPCRs. We have further shown that activation of AT₁R by Ang II increases 2-AG levels in CHO cells. These findings suggest that 2-AG is commonly released following activation of Ca²⁺-mobilizing GPCRs and serves as a paracrine signal to activate CB₁R in neighboring cells.     

Dimerization with Cannabinoid Receptors Allosterically Modulates Delta Opioid Receptor Activity during Neuropathic Pain

The diversity of receptor signaling is increased by receptor heteromerization leading to dynamic regulation of receptor function. While a number of studies have demonstrated that family A G-protein-coupled receptors are capable of forming heteromers in vitro, the role of these heteromers in normal physiology and disease has been poorly explored. In this study, direct interactions between CB₁ cannabinoid and delta opioid receptors in the brain were examined. Additionally, regulation of heteromer levels and signaling in a rodent model of neuropathic pain was explored. First we examined changes in the expression, function and interaction of these receptors in the cerebral cortex of rats with a peripheral nerve lesion that resulted in neuropathic pain. We found that, following the peripheral nerve lesion, the expression of both cannabinoid type 1 receptor (CB₁R) and the delta opioid receptor (DOR) are increased in select brain regions. Concomitantly, an increase in CB₁R activity and decrease in DOR activity was observed. We hypothesize that this decrease in DOR activity could be due to heteromeric interactions between these two receptors. Using a CB₁R-DOR heteromer-specific antibody, we found increased levels of CB₁R-DOR heteromer protein in the cortex of neuropathic animals. We subsequently examined the functionality of these heteromers by testing whether low, non-signaling doses of CB₁R ligands influenced DOR signaling in the cortex. We found that, in cortical membranes from animals that experienced neuropathic pain, non-signaling doses of CB₁R ligands significantly enhanced DOR activity. Moreover, this activity is selectively blocked by a heteromer-specific antibody. Together, these results demonstrate an important role for CB₁R-DOR heteromers in altered cortical function of DOR during neuropathic pain. Moreover, they suggest the possibility that a novel heteromer-directed therapeutic strategy for enhancing DOR activity, could potentially be employed to reduce anxiety associated with chronic pain.     

Effect of postsynaptic blockade of neuromuscualar transmission on properties of the frog fast muscle fiber membrane

Sensitivity of the postsynpatic membrane to acetylcholine, the resting membrane potential, input resistance, and membrane time constant of fast muscle fibers were measured in experiments on frogs. Complete immobilization of the animals with D-tubocurarine or local immobilization of a muscle with α-bungarotoxin was found not to affect these parameters of the muscle membrane, whereas denervation of the muscle widens the zone of postsynaptic sensitivity to acetylcholine, lowers the resting membrane potential, and increases the input resistance and time constant of the muscle membrane. These results are evidence that neurotrophic control of the frog fast muscle fiber membrane is achieved mainly by substances reaching the muscle via axoplasmic transport and not by the character of the neuronal discharge and motor activity or by synaptic acetylcholine.     

Paxillin and focal adhesion kinase colocalise in human skeletal muscle and its associated microvasculature

Focal adhesion kinase (FAK) and paxillin are functionally linked hormonal- and mechano-sensitive proteins. We aimed to describe paxillin’s subcellular distribution using widefield and confocal immunofluorescence microscopy and test the hypothesis that FAK and paxillin colocalise in human skeletal muscle and its associated microvasculature. Percutaneous muscle biopsies were collected from the m. vastus lateralis of seven healthy males, and 5-μm cryosections were stained with anti-paxillin co-incubated with anti-dystrophin to identify the sarcolemma, anti-myosin heavy chain type I for fibre-type differentiation, anti-dihydropyridine receptor to identify T-tubules, lectin UEA-I to identify the endothelium of microvessels and anti-α-smooth muscle actin to identify vascular smooth muscle cells (VSMC). Colocalisation of anti-paxillin with anti-dystrophin or anti-FAK was quantified using Pearson’s correlation coefficient on confocal microscopy images. Paxillin was primarily present in (sub)sarcolemmal regions of skeletal muscle fibres where it colocalised with dystrophin (r = 0.414 ± 0.026). The (sub)sarcolemmal paxillin immunofluorescence intensity was ~2.4-fold higher than in sarcoplasmic regions (P < 0.001) with sarcoplasmic paxillin immunofluorescence intensity ~10 % higher in type I than in type II fibres (P < 0.01). In some longitudinally orientated fibres, paxillin formed striations that corresponded to the I-band region. Paxillin immunostaining was highest in endothelial and VSMC and distributed heterogeneously in both cell types. FAK and paxillin colocalised at (sub)sarcolemmal regions and within the microvasculature (r = 0.367 ± 0.036). The first images of paxillin in human skeletal muscle suggest paxillin is present in (sub)sarcolemmal and I-band regions of muscle fibres and within the microvascular endothelium and VSMC. Colocalisation of FAK and paxillin supports their suggested role in hormonal and mechano-sensitive signalling.     

Interaction Between Central Opioidergic and Glutamatergic Systems on Food Intake in Neonatal Chicks: Role of NMDA,AMPA and mGLU1 Receptors

The present study was designed to examine the role of opioidergic and glutamatergic systems on feeding behavior in neonatal meat-type chicken. In experiment 1, FD₃ neonatal broilers ICV injected with (A) saline, (B) DAMGO (µ-opioid receptor agonist, 125 pmol), (C) MK-801 (NMDA glutamate receptors antagonist, 15 nmol) and (D) combination of DAMGO plus MK-801. Experiments 2–5 were similar to experiment 1, except FD₃ chicks ICV injected with CNQX (AMPA glutamate receptors antagonist, 390 nmol), AIDA (mGLU₁ receptors antagonist, 2 nmol), LY341495 (mGLU₂ receptors antagonist, 150 nmol) and UBP1112 (mGLU₃ receptors antagonist, 2 nmol) instead of MK-801, respectively. In experiments 6–10, FD₃ chicks ICV injected as the same as procedure to the experiments 1–5, except to inject with DPDPE (δ-opioid receptor agonist, 40 nmol) instead of the DAMGO. The experiments 11–15 were similar to the experiments 1–5, except neonatal broilers ICV injected with U-50488H (κ-opioid receptor agonist, 30 nmol) instead of DAMGO. Then the cumulative food intake measured until 120 min post injection. According to the results, ICV injection of DAMGO, significantly decreased food intake (P?<?0.05) while DPDPE and U-50488H increased feeding behavior compared to the control group (P?<?0.05). Co-injection of the DAMGO?+?MK-801 and DAMGO?+?AIDA, significantly decreased DAMGO-induced hypophagia in neonatal chicks (P?<?0.05). Also, co-injection of the DPDPE?+?CNQX significantly amplified DPDPE induced feeding behavior (P?<?0.05). These results suggested interconnection between central opioidergic and glutamatergic systems on feeding behavior mediates via µ- and δ-opioid receptor with NMDA, AMPA and mGLU₁ receptors in FD₃ neonatal broilers. These findings may shed light on the circuitry underlying interconnection between central opioidergic and glutamatergic systems on feeding behavior.     

C. elegans Dopaminergic D2-Like Receptors Delimit Recurrent Cholinergic-Mediated Motor Programs during a Goal-Oriented Behavior

Caenorhabditis elegans male copulation requires coordinated temporal-spatial execution of different motor outputs. During mating, a cloacal circuit consisting of cholinergic sensory-motor neurons and sex muscles maintains the male''s position and executes copulatory spicule thrusts at his mate''s vulva. However, distinct signaling mechanisms that delimit these behaviors to their proper context are unclear. We found that dopamine (DA) signaling directs copulatory spicule insertion attempts to the hermaphrodite vulva by dampening spurious stimulus-independent sex muscle contractions. From pharmacology and genetic analyses, DA antagonizes stimulatory ACh signaling via the D2-like receptors, DOP-2 and DOP-3, and Gα_o/i proteins, GOA-1 and GPA-7. Calcium imaging and optogenetics suggest that heightened DA-expressing ray neuron activities coincide with the cholinergic cloacal ganglia function during spicule insertion attempts. D2-like receptor signaling also attenuates the excitability of additional mating circuits to reduce the duration of mating attempts with unproductive and/or inappropriate partners. This suggests that, during wild-type mating, simultaneous DA-ACh signaling modulates the activity threshold of repetitive motor programs, thus confining the behavior to the proper situational context.     

The Significance and Mechanism of Propofol on Treatment of Ischemia Reperfusion Induced Lung Injury in Rats

This study is aimed to investigate the efficacy and underlying the mechanism of propofol in treatment of ischemia reperfusion (IR)-induced lung injury in rats, providing a novel insight of therapeutic strategy for IR-induced lung injury. 120 healthy SD rats were selected and randomly divided into sham operation group, IR group, and propofol group (40 rats per group). Bronchoalveolar lavage fluid (BALF) protein content, serum protein content, lung permeability index, lung water content rate, methane dicarboxylic aldehyde (MDA) in lung tissue, superoxide dismutase (SOD), nitric oxide (NO), endothelin (ET-1), toll-like receptor 4 (TLR4), nuclear factor (NF-κB), and tumor necrosis factor-α (TNF-α) were examined and compared among different groups to evaluate the therapeutical effects of propofol on IR-induced lung injury and analyze the mechanism. In sham operation group, neither change in lung tissue nor pulmonary interstitial edema or alveolar wall damage was found under microscope; in IR group, marked pulmonary interstitial edema and alveolar wall damage complicated with inflammatory cell infiltration and hemorrhage were found; in propofol group, alveolar wall widening was observed, however, hemorrhage in alveolar cavity, inflammatory infiltration and tissue damage were less significant than in IR group. At 3 h after reperfusion, BALF protein content, lung permeability index, and lung water content rate were all significantly increased in IR group and propofol group, while the serum protein content was significantly lower than sham operation group (p < 0.05). Moreover, we found that the change of above parameters in propofol group was less significant than in IR group (p < 0.05). No statistically significant difference was found in ET-1 levels in different groups (p > 0.05). In contrast, MDA and NO in IR group and propofol group were significantly increased, while SOD activity was significantly decreased (p < 0.05). Furthermore, the change of above parameters in propofol group was less significant than in IR group (p < 0.05). In addition, mRNAs of TLR4, NF-κB, and TNF-α were significantly increased in IR group and propofol group (p < 0.05) with more significant change in IR group compared with propofol group (p < 0.05). Propofol has protective effects against IR-induced lung injury by improving activity of oxygen radical and restoring NO/ET-1 dynamic balance. Besides, regulation of TLR4, NF-κB, and TNF-α by propofol also play important role in alleviating IR-induced lung injury.     

Characterisation of the cannabinoid receptor system in synovial tissue and fluid in patients with osteoarthritis and rheumatoid arthritis

Introduction

Cannabis-based medicines have a number of therapeutic indications, including anti-inflammatory and analgesic effects. The endocannabinoid receptor system, including the cannabinoid receptor 1 (CB₁) and receptor 2 (CB₂) and the endocannabinoids, are implicated in a wide range of physiological and pathophysiological processes. Pre-clinical and clinical studies have demonstrated that cannabis-based drugs have therapeutic potential in inflammatory diseases, including rheumatoid arthritis (RA) and multiple sclerosis. The aim of this study was to determine whether the key elements of the endocannabinoid signalling system, which produces immunosuppression and analgesia, are expressed in the synovia of patients with osteoarthritis (OA) or RA.

Methods

Thirty-two OA and 13 RA patients undergoing total knee arthroplasty were included in this study. Clinical staging was conducted from x-rays scored according to Kellgren-Lawrence and Larsen scales, and synovitis of synovial biopsies was graded. Endocannabinoid levels were quantified in synovial fluid by liquid chromatography-mass spectrometry. The expression of CB₁ and CB₂ protein and RNA in synovial biopsies was investigated. Functional activity of these receptors was determined with mitogen-activated protein kinase assays. To assess the impact of OA and RA on this receptor system, levels of endocannabinoids in the synovial fluid of patients and non-inflamed healthy volunteers were compared. The activity of fatty acid amide hydrolase (FAAH), the predominant catabolic endocannabinoid enzyme, was measured in synovium.

Results

CB₁ and CB₂ protein and RNA were present in the synovia of OA and RA patients. Cannabinoid receptor stimulation of fibroblast-like cells from OA and RA patients produced a time-dependent phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2 which was significantly blocked by the CB₁ antagonist SR141716A. The endocannabinoids anandamide (AEA) and 2-arachidonyl glycerol (2-AG) were identified in the synovial fluid of OA and RA patients. However, neither AEA nor 2-AG was detected in synovial fluid from normal volunteers. FAAH was active in the synovia of OA and RA patients and was sensitive to inhibition by URB597 (3'-(aminocarbonyl) [1,1'-biphenyl]-3-yl)-cyclohexylcarbamate).

Conclusion

Our data predict that the cannabinoid receptor system present in the synovium may be an important therapeutic target for the treatment of pain and inflammation associated with OA and RA.