Cannabinoid receptor stimulation of guanosine-5′-O-(3-[35S]thio)triphosphate binding in rat brain membranes

Cannabinoid receptors belong to the class of G-protein-coupled receptors which inhibit adenylyl cyclase. Coupling of receptors to G-proteins can be assessed by the ability of agonists to stimulate guanosine-5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding in the presence of excess GDP. The present study examined the effect of cannabinoid agonists on [³⁵S]GTPγS binding in rat brain membranes. Assays were conducted with 0.05 nM [³⁵S]GTPγS, incubated with rat cerebellar membranes, 1–30 μM GDP and the cannabinoid agonist WIN 55212-2. Results showed that the ability of WIN 55212-2 to stimulate [³⁵S]GTPγS binding increased with increasing concentrations of GDP, with 10–30 μM GDP providing approximately 150–200% stimulation by the cannabinoid agonist. The pharmacology of cannabinoid agonist stimulation of [³⁵S]GTPγS binding paralleled that of previously reported receptor binding and adenylyl cyclase assays, and agonist stimulation of [³⁵S]GTPγS binding was blocked by the cannabinoid antagonist SR141716A. Brain regional studies revealed widespread stimulation of [³⁵S]GTPγS binding by WIN 55212-2 in a number of brain areas, consistent with in vitro [³⁵S]GTPγS autoradiography. These results demonstrate that [³⁵S]GTPγS binding in the presence of excess GDP is an effective measure of cannabinoid receptor coupling to G-proteins in brain membranes.     

Effects of a selective cannabinoid CB2 agonist and antagonist on intravenous nicotine self administration and reinstatement of nicotine seeking

Over the last decade there have been significant advances in the discovery and understanding of the cannabinoid system along with the development of pharmacologic tools that modulate its function. Characterization of the crosstalk between nicotine addiction and the cannabinoid system may have significant implications on our understanding of the neurobiological mechanisms underlying nicotine dependence. Two types of cannabinoid receptors (CB1 and CB2) have been identified. CB1 receptors are expressed in the brain and modulate drug taking and drug seeking for various drugs of abuse, including nicotine. CB2 receptors have been recently identified in the brain and have been proposed to play a functional role in mental disorders and drug addiction. Our objective was to explore the role of CB2 receptors on intravenous nicotine self administration under two schedules of reinforcement (fixed and progressive ratio) and on nicotine seeking induced by nicotine priming or by nicotine associated cues. For this, we evaluated the effects of various doses of the selective CB2 antagonist AM630 (1.25 to 5 mg/kg) and CB2 agonist AM1241 (1 to 10 mg/kg) on these behavioral responses in rats. Different groups of male Long Evans rats were trained to lever press for nicotine at a unit dose of 30 μg/kg/infusion. Subsequently, animals were randomized using a Latin-square design and injected with either AM1241 or AM630 using a counterbalanced within subject design. Administration of the CB2 ligands did not affect either nicotine-taking nicotine-seeking behavior. Our results do not support the involvement of CB2 receptors in nicotine-taking or nicotine-seeking behavior.     

Disruption of Potential α-Helix in the G Loop of the Guinea: Pig 5-Hydroxytryptamine2 Receptor Does Not Prevent Receptor Coupling to Phosphoinositide Hydrolysis

Abstract: Heterogeneity of the 5-hydroxytryptamine₂ (5-HT₂) receptor across species has been implicated in several pharmacological and physiological studies. Although 5-HT₂ receptors in the rat have been linked to increases in Phosphoinositide (PI) hydrolysis, little evidence exists to support the association of guinea pig 5-HT₂ receptors with Pl hydrolysis, the second messenger generally linked with 5-HT₂receptors. In the present study, we have taken a molecular and biochemical approach to determining whether species differences in brain 5-HT₂ receptors exist between rat and guinea pig. First, we isolated partial cortical 5-HT_a receptor cDNA clones that encompassed the third intracellular loop, a receptor area putatively important in receptor-effector coupling. The amino acid sequences deduced from the cDNA clones for rat and guinea pig brain 5-HT₂ receptor were 97% homologous. However, the guinea pig 5-HT₂ receptor had two tandem substitutions that disrupted a potential alpha helix in the region of the third cytoplasmic loop, which theoretically could alter the intracellular coupling of the guinea pig cortical 5-HT₂ receptor. Because of these molecular differences, we examined further the pharmacological activation of the brain 5-HT₂ receptor from guinea pig. 5-HT and the 5-HT₂ receptor agonist α-methyl-5-HT increased PI hydrolysis in guinea pig cortical slices whereas the 5-HT_1c receptor agonist 5-methyltryptamine was significantly less potent. In addition, the 5-HT₂ receptor antagonists LY53857, ketanserin, and spiperone blocked 5-HT-stimulated Pl hydrolysis. These pharmacological data suggested that activation of the 5-HT₂ receptor in guinea pig cortical slices was associated with PI hydrolysis. Thus, although areas of the guinea pig brain 5-HT₂ receptor that influence receptor-effector coupling were different from the rat, such differences were not critical to receptor-effector coupling because, as in the rat, guinea pig brain 5-HT₂ receptors were also coupled to PI hydrolysis.     

Cannabinoid receptor antagonists and fatty acids alter endocannabinoid system gene expression and COX activity

Cyclooxygenase (COX) possesses substrate affinity for the endocannabinoids (EC) anandamide (AEA) and 2-arachidonylglycerol (2-AG). We hypothesized that selective antagonism/activation of the cannabinoid receptors will increase COX activity and the availability of EC as substrates will lead to higher COX activity. Since the relationship between EC signaling of the endocannabinoid system (ECS) and the COX pathway in muscle has not been investigated, we examined agonist, antagonists and polyunsaturated fatty acid effects on ECS genes in myoblasts. At 50% confluency, C2C12 myoblasts were pretreated with 5 μM of the cannabinoid receptor (CB)2 inverse agonist AM630 for 2 h and one with both AM630 and 1 μM of the CB1 antagonist NESS0327. Cell cultures pretreated with AM630 were then administered with 25 μM of either arachidonic acid (20:4n6), eicosapentaenoate (EPA) (20:5n3), docosahexaenoate (DHA) (22:6n3), AEA or bovine serum albumin (vehicle control) for 24 h. Quantitative polymerase chain reaction analyses were performed looking at ECS and prostaglandin genes. Total COX activity and COX-1 protein were greater in the AM630+AEA-treated cells compared to all other cell cultures. The mRNA for the AEA synthesis enzyme N-acyl phosphatidylethanolamine phospholipase D and the 2-AG synthesis enzymes diacylglycerol lipase (DAGL)α and DAGLβ were higher in AM630+EPA-treated cells compared to the other groups. The mRNA levels of CB1 and CB2 were both highest in the AM630+EPA group. The mRNA for interleukin-6 and tumor necrosis factor-α was higher with AEA but lower with DHA and docosahexaenoyl ethanolamide (DHEA), supporting previous findings that the EC AEA supports activation of the COX system. These findings suggest that COX activity and protein levels are influenced by the ECS, specifically by the ligand AEA for CB1 and by inverse agonism of CB2.     

Autoradiography of Serotonin 5-HT1A Receptor-Activated G Proteins in Guinea Pig Brain Sections by Agonist-Stimulated [35S]GTPγS Binding

Abstract: G protein activation mediated by serotonin 5-HT_1A and 5-HT_1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [³⁵S]GTPγS binding to brain sections. [³⁵S]GTPγS binding was stimulated by the mixed 5-HT_1A/5-HT_1B/D agonist L694247 in brain structures enriched in 5-HT_1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [³⁵S]GTPγS binding in brain regions with high densities of 5-HT_1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [³⁵S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5-HT_1B inverse agonist SB224289 (10 µM) did not affect the L694247-mediated [³⁵S]GTPγS binding response, and the mixed 5-HT_1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [³⁵S]GTPγS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT_1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [³⁵S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [³⁵S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [³⁵S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT_1A but not 5-HT_1B/D receptors can be measured in guinea pig brain sections.     

Binding of [35S]GTPgammaS stimulated by (+)-pentazocine sigma receptor agonist, is abundant in the guinea pig spleen

Here we measured sigma receptor agonist, [3H](+)-pentazocine binding and (+)-pentazocine-stimulated [35S]GTPgammaS binding throughout brain regions and peripheral organs of mice and guinea pigs to investigate the distribution of G protein-coupled sigma receptors. There was no significant correlationship between both distributions, in which the [3H](+)-pentazocine binding is highest in the liver of each species, while the [35S]GTP-gammaS binding is highest in the guinea pig spleen. The agonist-stimulated [35S]GTPgammaS binding in the spleen was also confirmed by in situ autoradiography using sections. Thus it is suggested that there are at least two subtypes, metabotropic and nonmetabotropic sigma receptors, and the former ones are abundant in the guinea pig spleen.     

CB<Subscript>1</Subscript> Receptors Mediated Inhibition of ATP-Induced [Ca<Superscript>2+</Superscript>]i Increase in Cultured Rat Spinal Dorsal Horn Neurons

Spinal cannabinoid receptor 1 (CB₁R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB₁R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB₁ receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca²⁺ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca²⁺]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca²⁺]i increase in cultured DH neurons. ATP-evoked [Ca²⁺]i increase in DH neurons was blocked by chelating extracellular Ca²⁺ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca²⁺]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca²⁺]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca²⁺ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca²⁺ response was mimicked by ACEA (CB₁R agonist), but was not influenced by AM1241 (CB₂R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca²⁺ mobilization was blocked by AM251 (CB₁ receptor antagonist), but was not influenced by AM630 (CB₂ receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB₁R rather than CB₂R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB₁R on P2X-channel-induced Ca²⁺ mobilization.     

A peptide-like substance from pituitary that acts like morphine. I. Isolation.

A method is described for extraction from bovine pituitary glands of a substance that shows several properties characteristic of opiates. The material inhibits the twitch tension of the electrically stimulated guinea pig longitudinal muscle-myenteric plexus preparation and of the electrically stimulated mouse vas deferens. This inhibition is reversed and blocked by the opiate antagonist naloxone. The extract also inhibits binding of the opiate agonist etorphine and the opiate antagonist naloxone to stereospecific binding sites in synaptic membranes of guinea pig brain. The inhibition of naloxone binding is decreased by Na⁺ in the manner characteristic of opiate agonists. The physiologic role of the pituitary opioid remains to be investigated.     

Feeding induced by cannabinoids is mediated independently of the melanocortin system

Background

Cannabinoids, the active components of marijuana, stimulate appetite, and cannabinoid receptor-1 (CB1-R) antagonists suppress appetite and promote weight loss. Little is known about how CB1-R antagonists affect the central neurocircuitry, specifically the melanocortin system that regulates energy balance.

Methodology/Principal Findings

Here, we show that peripherally administered CB1-R antagonist (AM251) or agonist equally suppressed or stimulated feeding respectively in A^y , which lack a functional melanocortin system, and wildtype mice, demonstrating that cannabinoid effects on feeding do not require melanocortin circuitry. CB1-R antagonist or agonist administered into the ventral tegmental area (VTA) equally suppressed or stimulated feeding respectively, in both genotypes. In addition, peripheral and central cannabinoid administration similarly induced c-Fos activation in brain sites suggesting mediation via motivational dopaminergic circuitry. Amperometry-detected increases in evoked dopamine (DA) release by the CB1-R antagonist in nucleus accumbens slices indicates that AM251 modulates DA release from VTA terminals.

Conclusions/Significance

Our results demonstrate that the effects of cannabinoids on energy balance are independent of hypothalamic melanocortin circuitry and is primarily driven by the reward system.     

Alkamides and a neolignan from Echinacea purpurea roots and the interaction of alkamides with G-protein-coupled cannabinoid receptors

Multiple chromatographic separations of the CHCl₃-soluble extract of the roots of Echinacea purpurea led to the isolation of 19 compounds. Four natural products, three alkamides and nitidanin diisovalerianate, were identified, and five further compounds were detected for the first time in this species. Additionally, 10 known E. purpurea metabolites were isolated. The structures were determined by mass spectrometry and advanced 1D and 2D NMR techniques. The bioactivity of the isolated compounds was studied in [³⁵S]GTPγS-binding experiments performed on rat brain membrane preparations. Both partial and inverse agonist compounds for cannabinoid (CB1) receptors were identified among the metabolites, characterized by weak to moderate interactions with the G-protein signaling mechanisms. The G-protein-modulating activities of the Echinacea compounds are rather far from the full agonist effects seen with the CB1 receptor agonist reference compound arachidonyl-2′-chloroethylamide (ACEA). However, upon coadministration with ACEA, a number of them proved capable of inhibiting the stimulation of the pure agonist, thereby demonstrating cannabinoid receptor antagonist properties.     

Rat, Mouse, and Guinea Pig Brain Development and Microtubule Assembly

The development of in vitro microtubule assembly and of tubulin concentration have been studied during brain maturation in the mouse and the rat, two species which have postnatal brain development, and in one species which is mature at birth, the guinea pig. (a) The rate of tubulin assembly is very slow soon after birth in both the mouse and rat; it increases progressively with age until adulthood. In contrast, in the guinea pig this rate is maximal at birth and slower rates are seen only at foetal stages. (b) Postnatal changes in the lag period of assembly and in the minimal concentration of tubulin (Cc) required to obtain in vitro assembly are seen in the mouse and the rat; in contrast these parameters are constant at all postnatal stages in the guinea pig with longer lag periods and lower Cc values being seen only at foetal stages. (c) Maximal rates of assembly, minimal lag periods, and minimal Cc values are restored after addition of microtubule-associated proteins to foetal guinea pig or young mouse and rat preparations, suggesting that the difference in the kinetic parameters of assembly between these species depends on differences in the concentration or activity of these proteins. (d) Maximal tubulin concentrations are observed before birth in the guinea pig and approximately at day 10 in the rat and mouse. Lennon A. M. et al. Rat, mouse, and guinea pig brain development and microtubule assembly. J. Neurochem. 35, 804–813 (1980).     

Effect of mequitazine a non sedative antihistamine on brain H1 receptors

Marked species variations occured in the relative activity of mequitazine (10-[3-quinuclidinylmethyl]-phenothiazine) on the binding of [³H]mepyramine to brain cortical membranes. Mequitazine was as potent as promethazine in the mouse but about 6 times less effective than promethazine in the guinea pig and human. On the other hand in the guinea pig mequitazine was as potent as promethazine on [³H]mepyramine binding in a peripheral organ (lung). Although mequitazine did not displace [³H]mepyramine in vivo in the mouse and guinea pig, its brain concentration (measured by [³H]mequitazine) was largely sufficient and corresponds to 90% of inhibition in vitro. Moreover in the mouse the brain regional distribution of [³H]mequitazine was very different from that of [³H]mepyramine, highest level was obtained in the cerebellum and hypothalamus was the poorest region with mequitazine whereas the reverse was true with mepyramine. All these results could suggest that mequitazine possesses a greater affinity for peripheral H₁ receptors which could explain the absence of sedative side-effects of this potent H₁ antagonist.     

High Levels of Ascorbic Acid, Not Glutathione, in the CNS of Anoxia-Tolerant Reptiles Contrasted with Levels in Anoxia-Intolerant Species

Abstract: Ascorbic acid and glutathione (GSH) are antioxidants and free radical scavengers that provide the first line of defense against oxidative damage in the CNS. Using HPLC with electrochemical detection, we determined tissue contents of these antioxidants in brain and spinal cord in species with varying abilities to tolerate anoxia, including anoxia-tolerant pond and box turtles, moderately tolerant garter snakes, anoxia-intolerant clawed frogs (Xenopus laevis), and intolerant Long-Evans hooded rats. These data were compared with ascorbate and GSH levels in selected regions of guinea pig CNS, human cortex, and values from the literature. Ascorbate levels in turtles were typically 100% higher than those in rat. Cortex, olfactory bulb, and dorsal ventricular ridge had the highest content in turtle, 5–6 µmol g^?1 of tissue wet weight, which was twice that in rat cortex (2.82 ± 0.05 µmol g^?1) and threefold greater than in guinea pig cortex (1.71 ± 0.03 µmol g^?1). Regionally distinct levels (2–4 µmol g^?1) were found in turtle cerebellum, optic lobe, brainstem, and spinal cord, with a decreasing anterior-to-posterior gradient. Ascorbate was lowest in white matter (optic nerve) in each species. Snake cortex and brainstem had significantly higher ascorbate levels than in rat or guinea pig, although other regions had comparable or lower levels. Frog ascorbate was generally in an intermediate range between that in rat and guinea pig. In contrast to ascorbate, GSH levels in anoxia-tolerant turtles, 2–3 µmol g^?1 of tissue wet weight, were similar to those in mammalian or amphibian brain, with no consistent pattern associated with anoxia tolerance. GSH levels in pond turtle CNS were significantly higher (by 10–20%) than in rat for several regions but were generally lower than in guinea pig or frog. GSH in box turtle and snake CNS were the same or lower than in rat or guinea pig. The distribution GSH in the CNS also had a decreasing anterior-to-posterior gradient but with less variability than ascorbate; levels were similar in optic nerve, brainstem, and spinal cord. The paradoxically high levels of ascorbate in turtle brain, which has a lower rate of oxidative metabolism than mammalian, suggest that ascorbate is an essential cerebral antioxidant. High levels may have evolved to protect cells from oxidative damage when aerobic metabolism resumes after a hypoxic dive.     

Comparative aspects of nitrobenzylthioinosine and dipyridamole inhibition of adenosine accumulation in rat and guinea pig synaptoneurosomes

Dipyridamole (DPR) and nitrobenzylthioinosine (NBI) inhibition of adenosine accumulation in synaptoneurosomes derived from rat cerebral cortex, rat cerebellum, guinea pig cerebral cortex and guinea pig cerebellum was investigated. The inhibition of adenosine accumulation by NBI was observed to be distinctly biphasic in both guinea pig and rat synaptoneurosomes. Such biphasic inhibition consisted of a nM potency component to inhibition, accounting for 20–30% of the maximum inhibition, and a μM potency component, accounting for the remaining 70–80% maximum inhibition. Such an inhibitory profile contrasts sharply with that of DPR which appears monophasic, with a mean IC₅₀ of between 10⁻⁷ M and 10⁻⁶ M, in all rat and guinea pig synaptoneurosomes preparations studied.Further differences between the potency of NBI and DPR in inhibiting [³H]adenosine accumulation were also noted. DPR was more potent in inhibiting [³H]adenosine accumulation in guinea pig cerebellar synaptoneurosomes than in cerebral cortex synaptoneurosomes. In rat synaptoneurosomes, the reverse selectivity was observed. DPR was also 2–6 fold (depending on brain region of comparison) more potent in inhibiting adenosine accumulation in guinea pig synaptoneurosomes than in inhibiting such accumulation in rat synaptoneurosomes. In contrast, NBI was approximately equipotent in inhibiting adenosine accumulation in rat and guinea pig synaptoneurosomes. Additional binding studies using [³H]NBI are also reported. The data presented are entirely consistent with the hypotheses that (1) NBI and DPR bind to functionally relevant sites and (2) there are different populations of nucleoside transporters in mammalian brain.     

Regional distribution of the muscarinic cholinoceptor and acetylcholinesterase in guinea pig brain

The muscarinic receptors in membranes prepared from guinea pig brain were studied using a radiolabeled antagonist, [³H]quinuclidinyl benzilate (QNB). The apparent dissociation constant of the QNB-receptor complex (K _d ) was similar in all regions, but the concentration of receptors was highest in the striatum, cerebral cortex, and hippocampus and lowest in the cerebellum. Similar distributions have been reported for other species, although the concentration of receptors in guinea pig brain is higher than in other species. Acetylcholine inhibited QNB binding with a Hill coefficient of 0.4–0.6. The concentration of acetylcholine required to inhibit binding by 50% (I₅₀) was lowest in the brain stem and more than 10 times higher in the hippocampus. Similar results have been reported for mouse brain. The activity of acetylcholinesterase was highest in the striatum, where the concentration of muscarinic receptors is highest, but did not vary greatly in other brain regions.RMD was seconded to the University of Melbourne to undertake this study.     

Estrone sulfate sulfohydrolase in the developing brain and pituitary of rat, mouse and guinea pig

The pattern of estrone sulfate sulfohydrolase (estrogen sulfatase) development in the brain of rat, mouse and guinea pig has been established by assaying whole homogenates. Activity was measurable in each species from the fetal state to adulthood. Maximum brain content was reached at about 20 days of age in rat, 14 days in mouse and 15 days in guinea pig. A considerable decrease occurred between 14 days and adulthood in mouse and lesser decreases were seen in rat and guinea pig. The subcellular distribution of enzyme in rat and mouse brain appeared to change from the immature to the adult state. No major differences in enzyme activity occurred between the sexes at any age. Tissue concentration of enzyme in the hypothalamic-preoptic area of rat and mouse was similar to that in the remainder of the brain. In guinea pig the brain concentration was slightly lower than that of the hypothalamic-preoptic region. Sulfatase content of the pituitary was low in all 3 species but the tissue concentration was considerably higher than that of brain, particularly in rat and mouse. Apparent Km values for brain sulfatase were in the range 6-17 microM, with no striking sex difference. Apparent Km's for pituitary sulfatase of immature rat and guinea pig were similar to those for brain in the same animals but that for mouse pituitary (0.9 microM) was much lower. It is unlikely that brain or pituitary sulfatase is by itself, a major factor in making available potentially active estrogen for use during differential sex development in these species.     

Oxidative metabolism of chicken polymorphonuclear leucocytes during phagocytosis

Summary The oxidative response to phagocytosis by chicken polymorphonuclear leucocytes was investigated as compared to guinea pig polymorphonuclear leucocytes.The polymorphs from both species respond to phagocytosis with an increased oxygen consumption, an increased generation of O₂ ^– and H₂O₂, and an increased oxidation of glucose through the hexose monophosphate shunt. The rate of oxygen consumption, and generation of O₂ ^– and H₂O₂ by phagocytosing chicken polymorphonuclear leucocytes is considerably lower than with phagocytosing guinea pig polymorphonuclear leucocytes. By contrast, the extent of hexose monophosphate shunt stimulation in chicken polymorphs is comparable to that of guinea pig polymorphs. Evidence is presented suggesting that H₂O₂ is preferentially degraded in chicken cells through the glutathione cycle, whereas catalase and myeloperoxidase are the two main H₂O₂ degrading enzymes in guinea pig cells.The 20,000 g fraction of the postnuclear supernatant of chicken polymorphs contains a cyanide-insensitive NADPH oxidizing activity which is stimulated during phagocytosis. Similar properties for the NADPH oxidizing activity of guinea pig polymorphs have been previously reported.It is concluded that the metabolic burst of phagocytosing chicken polymorphonuclear leucocytes is qualitatively similar to that of guinea pig polymorphonuclear leucocytes, but the latter cells are more active in all the biochemical parameters that have been measured. The difference in the H₂O₂ degradation pathways between the two species is accounted for by the lack of myeloperoxidase and catalase in chicken polymorphs.     

A neutral CB1 receptor antagonist reduces weight gain in rat

Cannabinoid (CB)1 receptor inverse agonists inhibit food intake in animals and humans but also potentiate emesis. It is not clear whether these effects result from inverse agonist properties or from the blockade of endogenous cannabinoid signaling. Here, we examine the effect of a neutral CB1 antagonist, AM4113, on food intake, weight gain, and emesis. Neutral antagonist and binding properties were confirmed in HEK-293 cells transfected with human CB1 or CB2 receptors. AM4113 had no effect on forskolin-stimulated cAMP production at concentrations up to 630 nM. The Ki value of AM4113 (0.80 +/- 0.44 nM) in competitive binding assays with the CB1/2 agonist [3H]CP55,940 was 100-fold more selective for CB1 over CB2 receptors. We determined that AM4113 antagonized CB1 receptors in brain by blocking hypothermia induced by CP55,940. AM4113 (0-20 mg/kg) significantly reduced food intake and weight gain in rat. Compared with AM251, higher doses of AM4113 were needed to produce similar effects on food intake and body weight. Unlike AM251 (5 mg/kg), a highly anorectic dose of AM4113 (10 mg/kg) did not significantly potentiate vomiting induced by the emetic morphine-6-glucoronide. We show that a centrally active neutral CB1 receptor antagonist shares the appetite suppressant and weight loss effects of inverse agonists. If these compounds display similar properties in humans, they could be developed into a new class of antiobesity agents.