The endocannabinoid system protects rat glioma cells against HIV-1 Tat protein-induced cytotoxicity. Mechanism and regulation

Cannabinoids modulate nitric oxide (NO) levels in cells of the central nervous system. Here we studied the effect of cannabinoid CB(1) and CB(2) receptor agonists on the release of NO and cell toxicity induced by the human immuno-deficiency virus-1 Tat protein (HIV-1 Tat) in rat glioma C6 cells. The CB(1) and CB(2) agonist WIN 55,212-2 inhibited the expression of inducible NO synthase (iNOS) and NO release caused by treatment of C6 cells with HIV-1 Tat and interferon-gamma (IFN-gamma). The effect of WIN 55,212-2 was uniquely due to CB(1) receptors, as shown by experiments carried out with selective CB(1) and CB(2) receptor agonists and antagonists. CB(1) receptor stimulation also inhibited HIV-1 Tat + IFN-gamma-induced and NO-mediated cell toxicity. Moreover, cell treatment with HIV-1 Tat + IFN-gamma induced a significant inhibition of CB(1), but not CB(2), receptor expression. This effect was mimicked by the NO donor GSNO, suggesting that the inhibition of CB(1) expression was due to HIV-1 Tat + IFN-gamma-induced NO overexpression. HIV-1 Tat + IFN-gamma treatment also induced a significant inhibition of the uptake of the endocannabinoid anandamide by C6 cells with no effect on anandamide hydrolysis. These findings show that the endocannabinoid system, through the modulation of the l-arginine/NO pathway, reduces HIV-1 Tat-induced cytotoxicity, and is itself regulated by HIV-1 Tat.     

Prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 and carbon monoxide reduces extracellular glutamate levels in primary rat cerebral cortex cell cultures

The effects of prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 (0.5 mg/kg s.c.), alone or in combination with carbon monoxide, on extracellular glutamate levels in primary rat cerebral cortical neuronal cultures, were investigated. Dam weight gain, pregnancy length and litter size at birth were not affected by prenatal treatment with WIN 55,212-2 and carbon monoxide alone or in combination. Basal and K(+)-evoked extracellular glutamate levels were reduced in cortical cultures from pups born to mothers exposed to WIN 55,212-2 and carbon monoxide alone or in combination compared to cultures from rats born to vehicle-treated mothers. In cultures obtained from rats exposed to vehicle or carbon monoxide alone during gestation, WIN 55,212-2 (0.01-100 nM) increased extracellular glutamate levels, displaying a bell-shaped concentration-response curve. In cultures from rats born to mothers exposed to WIN 55,212-2 alone or in combination with carbon monoxide the WIN 55,212-2 ( 1 nM)-induced increase in extracellular glutamate levels was lower than that observed in cultures from rats born to vehicle-treated mothers and similar at those observed at 10 and 100 nM concentrations. The selective CB1 receptor antagonist SR141716A (10 nM) counteracted the WIN 55,212-2-induced increase in extracellular glutamate levels in cultures exposed to vehicle or carbon monoxide during gestation, but failed to antagonise it in cultures from rats born to mothers exposed to WIN 55,212-2 alone or in combination with carbon monoxide. These findings provide evidence that prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 and carbon monoxide, alone or in combination, is associated with an impairment in cortical glutamatergic transmission. It could be speculated that such detrimental effects might be involved in the reported deficit in learning and memory associated with prenatal marijuana exposure.     

Novel, not adenylyl cyclase-coupled cannabinoid binding site in cerebellum of mice

In this study we report data suggesting the presence of a non-CB1, non-CB2 cannabinoid site in the cerebellum of CB1-/- mice. We have carried out [(35)S]GTPgammaS binding experiments in striata, hippocampi, and cerebella of CB1-/- and CB1(+/+) mice with Delta(9)-THC, WIN55,212-2, HU-210, SR141716A, and SR144528. In CB1-/- mice Delta(9)-THC and HU-210 did not stimulate [(35)S]GTPgammaS binding. However, WIN55,212-2 was able to stimulate [(35)S]GTPgammaS binding in cerebella of CB1-/- mice. The maximal effect of this stimulation was 31% that of wild type animals. This effect was reversible neither by CB1 nor CB2 receptor antagonists. Similar results were obtained with the endogenous cannabinoid, anandamide. However, adenylyl cyclase was not inhibited by WIN55,212-2 or anandamide in the CB1(minus sign/minus sign) animals. In striata and hippocampi of CB1-/- mice no [(35)S]GTPgammaS stimulation curve could be obtained with WIN55,212. Our findings suggest that there is a non-CB1 non-CB2 receptor present in the cerebellum of CB1-/- mice.     

CB1-cannabinoid receptors are involved in the modulation of non-synaptic [3H]serotonin release from the rat hippocampus

In the present study we investigated whether serotonin release in the hippocampus is subject to regulation via cannabinoid receptors. Both rat and mouse hippocampal slices were preincubated with [3H]serotonin ([3H]5-HT) and superfused with medium containing serotonin reuptake inhibitor citalopram hydrobromide (300 nM). The cannabinoid receptor agonist R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate (WIN55,212-2, 1 microM) did not affect either the resting or the electrically evoked [3H]5-HT release. In the presence of the ionotropic glutamate receptor antagonists D(-)-2-amino-5-phosphonopentanoic acid (AP-5, 50 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione-disodium (CNQX, 10 microM) the evoked [3H]5-HT release was decreased significantly. Similar findings were obtained when CNQX (10 microM) was applied alone with WIN55,212-2. This effect was abolished by the selective cannabinoid receptor subtype 1 (CB1) antagonists N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716, 1 microM) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide trifluoroacetate salt (AM251, 1 microM). Similarly to that observed in rats, WIN55,212-2 (1 microM) decreased the evoked [3H]5-HT efflux in wild-type mice (CB1+/+). The inhibitory effect of WIN55,212-2 (1 microM) was completely absent in hippocampal slices derived from mice genetically deficient in CB1 cannabinoid receptors (CB1-/-). Relatively selective degeneration of fine serotonergic axons by the neurotoxin parachloramphetamine (PCA) reduced significantly the tritium uptake and the evoked [3H]5-HT release. In addition, PCA, eliminated the effect of WIN55,212-2 (1 microM) on the stimulation-evoked [3H]5-HT efflux. In contrast to the PCA-treated animals, WIN55,212-2 (1 microM) reduced the [3H]5-HT efflux in the saline-treated group. Our data suggest that a subpopulation of non-synaptic serotonergic afferents express CB1 receptors and activation of these CB1 receptors leads to a decrease in 5-HT release.     

Modulation of cannabinoid agonist binding by 5-HT in the rat cerebellum

Cross-talk between cannabinoid CB1 and serotonin 5-HT receptors in rat cerebellar membranes was investigated using radioligand binding. In competition against the CB1 antagonist, [3 H]SR141716A, the agonist, WIN 55,212-2 yielded a biphasic isotherm. The majority of binding was to a high-affinity state that was significantly reduced by the GTP analogue, Gpp(NH)p. Interestingly, 5-HT enhanced the high-affinity binding constant of WIN 55,212-2 while attenuating the proportion of high-affinity binding. 5-HT also significantly reduced the proportion of high-affinity binding of the cannabinoid agonist, HU 210, but had no effect on the agonist, CP 55,940. The effect of 5-HT on WIN 55,212-2 binding was inhibited by the 5-HT2 receptor antagonist ritanserin as well as Gpp(NH)p, suggesting a dependence on the 5-HT2 receptor and on G protein-receptor interactions, respectively. Subsequent [3 H]WIN 55,212-2 dissociation kinetic experiments revealed that 5-HT promoted a slower-dissociating species of radiolabelled agonist-receptor complex. Our findings support a membrane-delimited cross-talk between two G protein-coupled receptors that are co-localized in certain cells of the central nervous system. Intriguingly, the cannabinoid agonist dependence of the 5-HT modulatory effect suggests that agonist-specific conformations of the CB1 receptor may also be important in determining the extent of this cross-talk.     

Inducible nitric oxide synthase knockout mouse macrophages disclose prooxidant effect of interferon-gamma on low-density lipoprotein oxidation.

To test our hypothesis that interferon-gamma (IFN-gamma) has a direct prooxidant effect on macrophage-mediated LDL oxidation behind its antioxidant effect via induction of inducible nitric oxide synthase (iNOS), we incubated LDL with wild-type (iNOS(+/+)) or iNOS knockout mouse (iNOS(-/-)) macrophages preincubated with IFN-gamma or IFN-gamma plus lipopolysaccharide (IFN-gamma/LPS) for 24 h. LDL oxidation was measured in terms of formation of thiobarbituric acid reactive substances (TBARS) and electrophoretic mobility. Thiol production, nitrite production, and superoxide production from macrophages were measured by using Ellman's assay, the Griess reagent, and the SOD-inhibitable cytochrome c reduction method, respectively. IFN-gamma alone or combined with LPS induced iNOS expression and increased nitrite production in iNOS(+/+) macrophages, but not in iNOS(-/-) macrophages. TBARS formation from LDL was suppressed in IFN-gamma- and IFN-gamma/LPS-treated iNOS(+/+) macrophages but was increased in IFN-gamma-treated iNOS(-/-) macrophages. In the presence of N(G)-monomethyl-l-arginine (l-NMMA), a NOS inhibitor, the suppressive effect of IFN-gamma and IFN-gamma/LPS was abolished and TBARS formation was even increased to a level above that of untreated iNOS(+/+) macrophage. NOC 18, an NO donor, dose dependently inhibited macrophage-mediated LDL oxidation. IFN-gamma increased superoxide and thiol productions in both types of macrophages. We conclude that IFN-gamma promotes macrophage-mediated LDL oxidation by stimulating superoxide and thiol production under conditions where iNOS-catalyzed NO release is restricted.     

Analogues and homologues of N-palmitoylethanolamide, a putative endogenous CB(2) cannabinoid, as potential ligands for the cannabinoid receptors.

The presence of CB(2) receptors was reported in the rat basophilic cell line RBL-2H3 and N-palmitoylethanolamide was proposed as an endogenous, potent agonist of this receptor. We synthesized a series of 10 N-palmitoylethanolamide homologues and analogues, varying by the elongation of the fatty acid chain from caproyl to stearoyl and by the nature of the amide substituent, respectively, and evaluated the affinity of these compounds to cannabinoid receptors in the rat spleen, RBL-2H3 cells and CHO-CB(1) and CHO-CB(2) receptor-transfected cells. In rat spleen slices, CB(2) receptors were the predominant form of the cannabinoid receptors. No binding of [(3)H]SR141716A was observed. [(3)H]CP-55,940 binding was displaced by WIN 55,212-2 and anandamide. No displacement of [(3)H]CP-55,940 or [(3)H]WIN 55,212-2 by palmitoylethanolamide derivatives was observed in rat spleen slices. In RBL-2H3 cells, no binding of [(3)H]CP-55,940 or [(3)H]WIN 55,212-2 could be observed and conversely, no inhibitory activity of N-palmitoylethanolamide derivatives and analogues was measurable. These compounds do not recognize the human CB(1) and CB(2) receptors expressed in CHO cells. In conclusion, N-palmitoylethanolamide was, in our preparations, a weak ligand while its synthesized homologues or analogues were essentially inactive. Therefore, it seems unlikely that N-palmitoylethanolamide is an endogenous agonist of the CB(2) receptors but it may be a compound with potential therapeutic applications since it may act via other mechanisms than cannabinoid CB(1)-CB(2) receptor interactions.     

Cannabinoid modulation of electrically evoked pH and oxygen transients in the nucleus accumbens of awake rats

Cannabinoid receptors have been implicated in the regulation of blood flow in the cerebral vasculature. Because the nucleus accumbens (NAc) shows high levels of central cannabinoid receptor 1 (CB1) expression we examined the effects of cannabinoids on the local transient alkaline shifts and increases in extracellular oxygen induced by electrical stimulation of the medial forebrain bundle (MFB) in conscious animals. These changes result from increases in cerebral blood flow (CBF) and metabolism in the NAc that are evoked by the stimulation. Oxygen and pH changes were monitored using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in the NAc of freely moving rats. Administration of the cannabinoid receptor agonist WIN55,212-2 potently inhibited extracellular oxygen and pH changes, an effect that was reversed and prevented by pre-treatment with the CB1 receptor antagonists SR141716A and AM251. The effects on pH following WIN55,212-2 were similar to those following nimodipine, a recognized vasodilator. When AM251 was injected alone, the amplitude of electrically evoked pH shifts was unaffected. Administration of AM404 and VDM11, endocannabinoid transport inhibitors, partially inhibited pH transients in a CB1 receptor-dependent manner. The present findings suggest that CB1 receptor activation modulates changes in two well-established indices of local blood flow and metabolism resulting from electrically evoked activation of ascending fibers. Although endogenous cannabinoid tone alone is not sufficient to modify these responses, uptake blockade demonstrates that the system has the potential to exert CB1-specific effects similar to those of full agonists.     

Cannabinoid-serotonin interactions in alcohol-preferring vs. alcohol-avoiding mice

Because cannabinoid and serotonin (5-HT) systems have been proposed to play an important role in drug craving, we investigated whether cannabinoid 1 (CB1) and 5-HT(1A) receptor ligands could affect voluntary alcohol intake in two mouse strains, C57BL/6 J and DBA/2 J, with marked differences in native alcohol preference. When offered progressively (3-10% ethanol) in drinking water, in a free-choice procedure, alcohol intake was markedly lower (approximately 70%) in DBA/2 J than in C57BL/6 J mice. In DBA/2 J mice, chronic treatment with the cannabinoid receptor agonist WIN 55,212-2 increased alcohol intake. WIN 55,212-2 effect was prevented by concomitant, chronic CB1 receptor blockade by rimonabant or chronic 5-HT(1A) receptor stimulation by 8-hydroxy-2-(di-n-propylamino)-tetralin, which, on their own, did not affect alcohol intake. In C57BL/6 J mice, chronic treatment with WIN 55,212-2 had no effect but chronic CB1 receptor blockade or chronic 5-HT(1A) receptor stimulation significantly decreased alcohol intake. Parallel autoradiographic investigations showed that chronic treatment with WIN 55,212-2 significantly decreased 5-HT(1A)-mediated [35S]guanosine triphosphate-gamma-S binding in the hippocampus of both mouse strains. Conversely, chronic rimonabant increased this binding in C57BL/6 J mice. These results show that cannabinoid neurotransmission can exert a permissive control on alcohol intake, possibly through CB1-5-HT(1A) interactions. However, the differences between C57BL/6 J and DBA/2 J mice indicate that such modulations of alcohol intake are under genetic control.     

Lipopolysaccharide-induced dopaminergic cell death in rat midbrain slice cultures: role of inducible nitric oxide synthase and protection by indomethacin

Glial cell activation associated with inflammatory reaction may contribute to pathogenic processes of neurodegenerative disorders, through production of several cytotoxic molecules. We investigated the consequences of glial activation by interferon-gamma (IFN-gamma)/lipopolysaccharide (LPS) in rat midbrain slice cultures. Application of IFN-gamma followed by LPS caused dopaminergic cell death and accompanying increases in nitrite production and lactate dehydrogenase release. Aminoguanidine, an inhibitor of inducible nitric oxide synthase (iNOS), or SB203580, an inhibitor of p38 mitogen-activated protein kinase, prevented dopaminergic cell loss as well as nitrite production. SB203580 also suppressed expression of iNOS and cyclooxygenase-2 (COX-2) induced by IFN-gamma/LPS. A COX inhibitor indomethacin protected dopaminergic neurons from IFN-gamma/LPS-induced injury, whereas selective COX-2 inhibitors such as NS-398 and nimesulide did not. Notably, indomethacin was able to attenuate neurotoxicity of a nitric oxide (NO) donor. Neutralizing antibodies against tumour necrosis factor-alpha and interleukin-1beta did not inhibit dopaminergic cell death caused by IFN-gamma/LPS, although combined application of these antibodies blocked lactate dehydrogenase release and decrease in the number of non-dopaminergic neurons. These results indicate that iNOS-derived NO plays a crucial role in IFN-gamma/LPS-induced dopaminergic cell death, and that indomethacin exerts protective effect by mechanisms probably related to NO neurotoxicity rather than through COX inhibition.     

CB1-independent inhibition of dopamine transporter activity by cannabinoids in mouse dorsal striatum

Cannabinoid drugs are known to affect dopaminergic neurotransmission in the basal ganglia circuitry. In this study, we used in vitro and in vivo techniques to investigate whether cannabinoid agonists and antagonist could affect dopaminergic transmission in the striatum by acting at the dopamine transporter. Incubation of striatal synaptosomes with the cannabinoid agonists WIN55,212-2 or methanandamide decreased dopamine uptake (IC(50) = 2.0 micromol/L and 3.1 micromol/L, respectively). A similar inhibitory effect was observed after application of the inactive WIN55,212-2 isomer, S(-)WIN55,212-3. The CB(1) antagonist AM251 did not reverse WIN55,212-2 effect but rather mimicked it. WIN55,212-2 and AM251 partially displaced the binding of the cocaine analog [(3)H]WIN35,428, thus acting as dopamine transporter pseudo-substrates in the high micromolar range. High-speed chronoamperometry measurements showed that WIN55,212-2 (4 mg/kg, i.p.) caused significant release of endogenous dopamine via activation of CB(1) receptors, followed by a reduction of dopamine clearance. This reduction was CB(1)-independent, as it was mimicked by S(-)WIN55,212-3. Administration of AM251 (1 and 4 mg/kg, i.p.) increased the signal amplitude and reduced the clearance of dopamine pressure ejected into the striatum. These results indicate that both cannabinoid agonists and antagonists inhibit dopamine transporter activity via molecular targets other than CB(1) receptors.     

Cannabinoids, hippocampal function and memory.

Prior studies from this laboratory have shown that the psychoactive ingredient in marijuana, delta9-tetrahydrocannabinol (THC), interferes with short-term memory (1-3) in both delayed match and nonmatch to sample tasks (DMS/DNMS). Recent experiments have shown that other cannabinoids such as the potent CB1 receptor agonist, WIN 55,212-2 produces a delay-dependent deficit in the DNMS task at a dose range (0.10-0.50 mg/kg) well below that of delta9-THC which was blocked by the CB11 receptor antagonist SR141716A (Sanofi Inc). The effects of WIN 55,212-2 at low doses were similar to those of isolated lesions of the hippocampus, whereas high doses (0.50 mg/kg, i.p.) produced effects similar to lesions of both hippocampus and surrounding retrohippocampal areas. The low dose effect was delay-dependent while the high dose introduced an additional deficit at short delays that was sensitive to both SR141716A and the GABA(B) receptor antagonist, phaclofen. Comparison of lesion vs. cannabinoid effects on DNMS performance suggests that CB1 receptors on hippocampal neurons interfere with the processing of DNMS task-specific information within a trial. CB1 receptors on hippocampal GABAergic interneurons and in retrohippocampal areas appear to influence the ability to maintain segregation of information between trials in the task.     

Cannabinoids Inhibit Acid-Sensing Ion Channel Currents in Rat Dorsal Root Ganglion Neurons

Local acidosis has been found in various pain-generating conditions such as inflammation and tissue injury. Cannabinoids exert a powerful inhibitory control over pain initiation via peripheral cognate receptors. However, the peripheral molecular targets responsible for the antinociceptive effects of cannabinoids are still poorly understood. Here, we have found that WIN55,212-2, a cannabinoid receptor agonist, inhibits the activity of native acid-sensing ion channels (ASICs) in rat dorsal root ganglion (DRG) neurons. WIN55,212-2 dose-dependently inhibited proton-gated currents mediated by ASICs. WIN55,212-2 shifted the proton concentration–response curve downwards, with an decrease of 48.6±3.7% in the maximum current response but with no significant change in the EC₅₀ value. The inhibition of proton-gated current induced by WIN55,212-2 was almost completely blocked by the selective CB1 receptor antagonist AM 281, but not by the CB2 receptor antagonist AM630. Pretreatment of forskolin, an AC activator, and the addition of cAMP also reversed the inhibition of WIN55,212-2. Moreover, WIN55,212-2 altered acid-evoked excitability of rat DRG neurons and decreased the number of action potentials induced by acid stimuli. Finally, WIN55,212-2 attenuated nociceptive responses to injection of acetic acid in rats. These results suggest that WIN55,212-2 inhibits the activity of ASICs via CB1 receptor and cAMP dependent pathway in rat primary sensory neurons. Thus, cannabinoids can exert their analgesic action by interaction with ASICs in the primary afferent neurons, which was novel analgesic mechanism of cannabinoids.     

Noradrenaline release-inhibiting receptors on PC12 cells devoid of alpha(2(-)) and CB(1) receptors: similarities to presynaptic imidazoline and edg receptors.

The aim of the present study was to classify release-inhibiting receptors on rat pheochromocytoma PC12 cells. Veratridine-evoked [3H]noradrenaline release from PC12 cells was inhibited by micromolar concentrations of the imidazoline and guanidine derivatives cirazoline, clonidine, aganodine, 1,3-di(2-tolyl)guanidine, BDF6143 and agmatine, and of the cannabinoid receptor agonist WIN55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-yl](1-naphthalenyl)methanone mesylate), but not by noradrenaline. The inhibitory effect of clonidine was antagonized by micromolar concentrations of rauwolscine and SR141716A (N-[piperidin-1-yl]-5-[4-chlorophenyl]-1-[2,4-dichlorophenyl]-4-methyl-1H-pyrazole-3-carboxamide). The potencies of the agonists and antagonists were compatible with an action at previously characterized presynaptic imidazoline receptors. 1-Oleoyl-lysophosphatidic acid, but not sphingosine-1-phosphate, produced an inhibition of release that was antagonized by 30 microM rauwolscine, 1 microM SR141716A and 10 microM LY320135 as well as by pretreatment of the cells with 100 microM clonidine for 72 h. Polymerase chain reaction (PCR) experiments on cDNA from PC12 mRNA suggest mRNA expression of lysophospholipid receptors encoded by the genes edg2, edg3, edg5 and edg7, but not of receptors encoded by edg1, edg4, edg6 and edg8, and not of alpha(2A(-))nd CB(1) receptors. In conclusion, PC12 cells are not endowed with alpha(2)-adrenoceptors and CB(1) cannabinoid receptors, but with an inhibitory receptor recognizing imidazolines, guanidines and WIN55,212-2 similar to that on sympathetic nerves. The PCR results and the ability of 1-oleoyl-LPA to mimic these drugs (also with respect to their susceptibility to antagonists) suggest that the release-inhibiting receptor may be an edg-encoded lysophospholipid receptor.     

Role of receptor internalization in the agonist-induced desensitization of cannabinoid type 1 receptors

Agonist-induced internalization of G protein-coupled receptors (GPCRs) is an important mechanism for regulating signaling transduction of functional receptors at the plasma membrane. We demonstrate here that both caveolae/lipid-rafts- and clathrin-coated-pits-mediated pathways were involved in agonist-induced endocytosis of the cannabinoid type 1 receptor (CB1R) in stably transfected human embryonic kidney (HEK) 293 cells and that the internalized receptors were predominantly sorted into recycling pathway for reactivation. The treatment of CB1 receptors with the low endocytotic agonist Δ⁹-THC induced a faster receptor desensitization and slower resensitization than the high endocytotic agonist WIN 55,212-2. In addition, the blockade of receptor endocytosis or recycling pathway markedly enhanced agonist-induced CB1 receptor desensitization. Furthermore, co-expression of phospholipase D2, an enhancer of receptor endocytosis, reduced CB1 receptor desensitization, whereas co-expression of a phospholipase D2 negative mutant significantly increased the desensitization after WIN 55,212-2 treatment. These findings provide evidences for the importance of receptor endocytosis in counteracting CB1 receptor desensitization by facilitating receptor reactivation. Moreover, in primary cultured neurons, the low endocytotic agonist Δ⁹-THC or anandamide exhibited a greater desensitization of endogenous CB1 receptors than the high endocytotic agonist WIN 55,212-2, CP 55940 or 2-arachidonoyl glycerol, indicating that cannabinoids with high endocytotic efficacy might cause reduced development of cannabinoid tolerance to some kind cannabinoid-mediated effects.     

Blockade of cannabinoid CB(1) receptor function protects against in vivo disseminating brain damage following NMDA-induced excitotoxicity

The ability of cannabinoid CB(1) receptors to influence glutamatergic excitatory neurotransmission has fueled interest in how these receptors and their endogenous ligands may interact in conditions of excitotoxic insults. The present study characterized the impact of stimulated and inhibited CB(1) receptor function on NMDA-induced excitotoxicity. Neonatal (6-day-old) rat pups received a systemic injection of a mixed CB(1) /CB(2) receptor agonist (WIN55,212-2) or their respective antagonists (SR141716A for CB(1) and SR144528 for CB(2) ) prior to an unilateral intrastriatal microinjection of NMDA. The NMDA-induced excitotoxic damage in the ipsilateral forebrain was not influenced by agonist-stimulated CB(1) receptor function. In contrast, blockade of CB(1), but not CB(2), receptor activity evoked a robust neuroprotective response by reducing the infarct area and the number of cortical degenerating neurons. These results suggest a critical involvement of CB(1) receptor tonus on neuronal survival following NMDA receptor-induced excitotoxicity in vivo.     

Ionizing radiation potentiates the induction of nitric oxide synthase by interferon-gamma (Ifn-gamma) or Ifn-gamma and lipopolysaccharide in bnl cl.2 murine embryonic liver cells: role of hydrogen peroxide

The effects of ionizing irradiation on the nitric oxide (NO) production in murine embryonic liver cell line, BNL CL.2 cells, were investigated. Various doses (5-40 Gy) of radiation made BNL CL.2 cells responsive to interferon-gamma alone for the production of NO in a dose-dependent manner. Small amounts of lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) synergized with IFN-gamma in the production of NO from irradiated BNL CL.2 cells, even though LPS or TNF-alpha alone did not induce NO production from the same cells. Immunoblots showed parallel induction of inducible nitric oxide synthase (iNOS). NO production in irradiated BNL CL.2 cells by IFN-gamma or IFN-gamma plus LPS was decreased by the addition of catalase, suggesting that H(2)O(2) produced by ionizing irradiation primed the cells to trigger NO production in response to IFN-gamma or IFN-gamma plus LPS. Furthermore, the treatment of nongamma-irradiated BNL CL.2 cells with H(2)O(2) made the cells responsive to IFN-gamma or IFN-gamma plus LPS for the production of NO. This study shows that ionizing irradiation has the ability to induce iNOS gene expression in responsive to IFN-gamma via the formation of H(2)O(2) in BNL CL.2 murine embryonic liver cells.     

Inhibition of peristaltic activity by cannabinoids in the isolated distal colon of mouse.

The effects of the cannabinoid receptor agonist Win 55,212-2 and of the competitive cannabinoid receptor antagonist SR 141716A on the electrically-evoked peristalsis of isolated distal colon of mouse were studied. Intraluminal pressure, longitudinal displacement, ejected fluid volume and changes in morphology of external intestinal wall were simultaneously recorded in the pre-drug period and in presence of Win 55,212-2 alone or in combination with SR 141716A. In the pre-drug period (control), peristaltic activity was characterised by regular, monophasic waves and the intraluminal content propelled towards anterograde (oro-aboral) direction with a propulsion velocity of 1.25 +/- 0.1 mm x s(-1). Pressure and shortening waves showed a peak amplitude of 2.44 +/- 0.32 kPa and 1.8 +/- 0.72 mm, respectively. The mean amount of fluid volume ejected during each contraction was 80 +/- 12.6 microl. The addition of Win 55,212-2 [10(-7)-10(-4) M] to the organ bath determined a dose-related attenuation of peristaltic activity consequent to the decrease of circular and longitudinal muscle strength. The decrease of contractile activity was followed by dose-dependent decrease of the amount of fluid ejected during peristalsis. The effects of Win 55,212-2 [10(-7)-10(-5) M] were prevented by SR 141716A, indicating the presence of cannabinoid CB1 receptors in the mouse distal colon. SR 141716A alone enhanced both tonic and phasic motor activities in the colonic longitudinal smooth muscle, suggesting that CB1 receptor antagonists could act either through antagonising the effect of endogenous CB1 receptor agonist or by an agonist effect on these receptors. The present results further support the hypothesis that cannabinoids perform a neuromodulatory role in various tracts of gastrointestinal system and first demonstrate their action also in the distal colon of rodents.