Effects of water-soluble cigarette smoke extracts upon the release of beta-hexosaminidase from RBL-2H3 basophilic leukaemia cells in response to substance P, compound 48/80, concanavalin A and antigen stimulation.

OBJECTIVE AND DESIGN: To determine whether water-soluble constituents of cigarette smoke affect mast cell function using an in vitro model, RBL-2H3 basophilic leukaemia cells. MATERIALS AND METHODS: RBL-2H3 cells were induced to degranulate in response to compound 48/80 and substance P, as assessed by monitoring the release of the granular enzyme beta-hexosaminidase, by treatment for 7 days with 20 microM quercetin. Responses to concanavalin A and antigen were determined by measuring the beta-hexosaminidase release from cells cultured on fibronectin-coated plates. RESULTS: The beta-hexosaminidase release response to compound 48/80 induced by quercetin treatment was accompanied by a release of lactate dehydrogenase, suggesting that degranulation is not the only process triggered by compound 48/80 under these conditions. Quercetin treatment reduced the beta-hexosaminidase release response to concanavalin A. Precoating of the culture wells with rat fibronectin enhanced the beta-hexosaminidase response to calcimycin, but not to concanavalin A. Under these conditions, concanavalin A did not induce a release of lactate dehydrogenase. The responses to c48/80, substance P, calcimycin, concanavalin A and antigen (after IgE pretreatment) were reduced by treatment with cigarette smoke solution obtained from standard and low-tar cigarettes (IR3 and IR5F). The effect of cigarette smoke solution from IR5F cigarettes upon the beta-hexosaminidase release elicited by compound 48/80 (in quercetin-treated cells) and by concanavalin A (in cells cultured on fibronectin-coated wells) could be prevented by N-acetyl-L-cysteine, but not with either hemoglobin, alpha-tocopherol, catalase or palmitoylethanolamide. N-acetyl-L-cysteine also reduced the effect of cigarette smoke solution upon the degranulation response to antigen. CONCLUSIONS: Under the conditions used, oxidants present in cigarette smoke solution from IR5F cigarettes reduce the ability of RBL-2H3 cells to degranulate in response to both immunological and non-immunological stimuli.

Fatty acid amide hydrolase: biochemistry, pharmacology, and therapeutic possibilities for an enzyme hydrolyzing anandamide, 2-arachidonoylglycerol, palmitoylethanolamide, and oleamide.

Fatty acid amide hydrolase (FAAH) is responsible for the hydrolysis of a number of important endogenous fatty acid amides, including the endogenous cannabimimetic agent anandamide (AEA), the sleep-inducing compound oleamide, and the putative anti-inflammatory agent palmitoylethanolamide (PEA). In recent years, there have been great advances in our understanding of the biochemical and pharmacological properties of the enzyme. In this commentary, the structure and biochemical properties of FAAH and the development of potent and selective FAAH inhibitors are reviewed, together with a brief discussion on the therapeutic possibilities for such compounds in the treatment of inflammatory pain and ischaemic states.

Effects of homologues and analogues of palmitoylethanolamide upon the inactivation of the endocannabinoid anandamide.

1. The ability of a series of homologues and analogues of palmitoylethanolamide to inhibit the uptake and fatty acid amidohydrolase (FAAH)-catalysed hydrolysis of [(3)H]-anandamide ([(3)H]-AEA) has been investigated. 2. Palmitoylethanolamide and homologues with chain lengths from 12 - 18 carbon atoms inhibited rat brain [(3)H]-AEA metabolism with pI(50) values of approximately 5. Homologues with chain lengths < or = eight carbon atoms gave < 20% inhibition at 100 microM. 3. R-palmitoyl-(2-methyl)ethanolamide, palmitoylisopropylamide and oleoylethanolamide inhibited [(3)H]-AEA metabolism with pI(50) values of 5.39 (competitive inhibition), 4.89 (mixed type inhibition) and 5.33 (mixed type inhibition), respectively. 4. With the exception of oleoylethanolamide, the compounds did not produce dramatic inhibition of [(3)H]-WIN 55,212-2 binding to human CB(2) receptors expressed on CHO cells. Palmitoylethanolamide, palmitoylisopropylamide and R-palmitoyl-(2-methyl)ethanolamide had modest effects upon [(3)H]-CP 55,940 binding to human CB(1) receptors expressed on CHO cells. 5. Most of the compounds had little effect upon the uptake of [(3)H]-AEA into C6 and/or RBL-2H3 cells. However, palmitoylcyclohexamide (100 microM) and palmitoylisopropylamide (30 and 100 microM) produced more inhibition of [(3)H]-AEA uptake than expected to result from inhibition of [(3)H]-AEA metabolism alone. 6. In intact C6 cells, palmitoylisopropylamide and oleoylethanolamide inhibited formation of [(3)H]-ethanolamine from [(3)H]-AEA to a similar extent as AM404, whereas palmitoylethanolamide, palmitoylcyclohexamide and R-palmitoyl-(2-methyl)ethanolamide were less effective. 7. These data provide useful information upon the ability of palmitoylethanolamide analogues to act as 'entourage' compounds. Palmitoylisopropylamide may prove useful as a template for design of compounds that reduce the cellular accumulation and metabolism of AEA without affecting either CB(1) or CB(2) receptors.

Effects of pH on the inhibition of fatty acid amidohydrolase by ibuprofen.

The pharmacological properties of fatty acid amidohydrolase (FAAH) at different assay pH values were investigated using [(3)H]-anandamide ([(3)H]-AEA) as substrate in rat brain homogenates and in COS-1 [corrected] cells transfected with wild type and mutant FAAH. Rat brain hydrolysis of [(3)H]-AEA showed pH dependency with an optimum around pH 8-9. Between pH 6.3 and 8.2, the difference in activity was due to differences in the V(max), rather than the K(M) values. For inhibition of rat brain [(3)H]-AEA metabolism by a series of known FAAH inhibitors, the potencies of the enantiomers of ibuprofen and phenylmethylsulphonyl fluoride (PMSF) were higher at pH 5.28 than at pH 8.37, whereas the reverse was true for oleyl trifluoromethylketone (OTMK) and arachidonoylserotonin. At both pH values, (-)ibuprofen was a mixed-type inhibitor of FAAH. The K(i)((slope)) and K(i)((intercept)) values for (-)ibuprofen at pH 5.28 were 11 and 143 microM, respectively. At pH 8.37, the corresponding values were 185 and 3950 microM, respectively. The pH dependency for the inhibition by OTMK and (-)ibuprofen was also seen in COS-1 [corrected] cells transiently transfected with either wild type, S152A or C249A FAAH. No differences in potencies between the wild type and mutant enzymes were seen. It is concluded that the pharmacological properties of FAAH are highly pH-dependent. The higher potency of ibuprofen at lower pH values raises the possibility that in certain types of inflamed tissue, the concentration of this compound following oral administration may be sufficient to inhibit FAAH.

Pharmacological properties of cannabinoid receptors in the avian brain: similarity of rat and chicken cannabinoid1 receptor recognition sites and expression of cannabinoid2 receptor-like immunoreactivity in the embryonic chick brain.

The pharmacological properties of brain cannabinoid receptors were investigated in brains of 35 day-old chickens, since little is known about the avian cannabinoid system. The cannabinoid1 receptor-selective antagonist ligand [3H]SR 141716A bound to chicken brain membranes with K(D) and Bmax values of 0.92+/-0.28 nM and 790+/-58 fmol/mg protein, respectively. The binding was inhibited by CP 55,940 with a pI50 value of 7.63+/-0.14 and by a series of compounds with the order of potency CP 55,940>R(+)WIN 55,212-2>R-1 methanandamide approximately DAK. S(-)WIN 55,212-3 and AM404 were without inhibitory effect at 1 microM. Similar results were found for rat brain membranes. For both rat and chicken brain membranes, addition of the non-hydrolysable GTP analogues Gpp[NH]p and GTPgammaS shifted the CP 55,940 inhibition curve to the right, consistent with an intact coupling to G-proteins in the preparations. Fatty acid amidohydrolase in chicken brain membranes was less sensitive to inhibition by phenylmethylsulphonyl fluoride and arachidonoyl serotonin than its rodent equivalent. However, when fatty acid amidohydrolase activity in the preparations was reduced by use of a lower assay membrane concentration, anandamide was found to inhibit the binding of [3H]SR 141716A to chicken membranes with a pI50 value of 6.39+/-0.16. Using a novel antibody raised to amino acids 346-359 from the C-terminal tail of the human cannabinoid2 receptor, it was found that embryonic chick brain tissue (and embryonic chick neurones in primary culture) expressed a approximately 53 kDa immunoreactive band. This immunoreactivity, which was prevented by preincubation of the antibody with the immunising peptide, was also seen in cells expressing the recombinant human cannabinoid, receptor, but was not seen in adult chicken brain homogenates or in rat cerebellar homogenates. However, a "classical" cannabinoid2-receptor component of [3H]WIN 55212-2 binding (i.e. a fraction inhibited by low concentrations of the cannabinoid2-receptor-selective antagonist SR 144528) was not found.

Serum-dependent effects of tamoxifen and cannabinoids upon C6 glioma cell viability.

In the present study, the effects of the combination of tamoxifen ((Z)-2[p-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylamine citrate) and three cannabinoids (Delta(9)-tetrahydrocannabinol [Delta(9)-THC], cannabidiol, and anandamide [AEA]) upon the viability of C6 rat glioma cells was assessed at different incubation times and using different culturing concentrations of foetal bovine serum (FBS). Consistent with previous data for human glioblastoma cells, the tamoxifen sensitivity of the cells was increased as the FBS content of the culture medium was reduced from 10 to 0.4 and 0%. The cells expressed protein kinase C alpha and calmodulin (the concentration of which did not change significantly as the FBS concentration was reduced), but did not express estrogen receptors. Delta(9)-THC and cannabidiol, but not AEA, produced a modest reduction in cell viability after 6 days of incubation in serum-free medium, whereas no effects were seen in 10% FBS-containing medium. There was no observed synergy between the effects of tamoxifen and the cannabinoids upon cell viability.

Differences in the pharmacological properties of rat and chicken brain fatty acid amidohydrolase.

The pharmacological properties of fatty acid amidohydrolase (FAAH) were investigated in brains of 35-day-old chickens, since nothing is known about the enzyme in avian species. FAAH activity towards both [(3)H]-palmitoylethanolamide (PEA) [K(M)=1.5 microM] and [(3)H]-anandamide (AEA) [K(M)=5.4 microM] was demonstrated in the chicken brains. The chicken FAAH was inhibited by the substrate analogues oleyl trifluoromethylketone (OTMK) and diazomethylarachidonyl ketone (DAK) with similar potencies to the rat FAAH. However, in contrast to the rat brain, phenylmethylsulphonyl fluoride (PMSF) and the enantiomers of ibuprofen had very weak effects on chicken brain FAAH. Indomethacin and niflumic acid were found to inhibit rat brain AEA hydrolysis. The inhibition by indomethacin was reversible and competitive, with a K(i) value of 120 microM. Chicken FAAH was less sensitive to indomethacin than its rodent counterpart, but the inhibition was also competitive (K(i)). It is concluded that chicken FAAH activity has different pharmacological properties to its rodent counterpart.

Neurotoxicity of glutamate in chick telencephalon neurons: reduction of toxicity by preincubation with carbachol, but not by the endogenous fatty acid amides anandamide and palmitoylethanolamide.

Exposure of chick telencephalon neurons in serum-free primary culture to glutamate produced a concentration-dependent cell toxicity as seen by an increase in lactate dehydrogenase (LDH) release that was blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine and was reduced by preincubation with the cholinergic agonist carbachol. Preincubation with a threshold concentration of NMDA did not prevent glutamate toxicity, suggesting that chick NMDA receptors do not desensitize in the manner reported for their rodent counterparts. Neither anandamide (arachidonyl ethanolamide, AEA) nor palmitoylethanolamide (PEA) was able to prevent the neurotoxicity produced by prolonged glutamate incubation, even under conditions in which the metabolism of the compounds by fatty acid amide hydrolase or AEA cellular uptake was blocked. It is concluded that treatments reported as granting neuroprotection towards glutamate toxicity in rodent primary neuronal cultures do not necessarily show the same properties in the chick.

Pharmacological properties of rat brain fatty acid amidohydrolase in different subcellular fractions using palmitoylethanolamide as substrate.

In the present study, the pharmacological properties of fatty acid amide hydrolase (FAAH) in subcellular fractions of rat brain were investigated using palmitoylethanolamide (PEA) and arachidonyl ethanolamide (anandamide, AEA) as substrates. FAAH hydrolysed [(3)H]PEA in crude homogenates with median K(m) and V(max) values of 2.9 microM and 2.14 nmol.(mg protein)(-1).min(-1), respectively. [(3)H]PEA hydrolysis was inhibited both by non-radioactive AEA (with a K(i) value very similar to the K(m) value for [(3)H]AEA as substrate using the same assay) and by R(-)ibuprofen (mixed-type inhibition K(i) and K'(i) values 88 and 720 microM, respectively). FAAH activity towards both [(3)H]PEA and [(3) myelin = cytosol, but there were no differences between the relative activities towards the two substrates in any of the fractions. [(3)H]PEA hydrolysis in mitochondrial, myelin, microsomal, and synaptosomal fractions was inhibited by oleyl trifluoromethylketone, phenylmethylsulphonyl fluoride, and the R(-)- and S(+)-enantiomers of the nonsteroidal anti-inflammatory drug ibuprofen, with mean IC(50) values in the ranges 0.028-0.041, 0.37-0.52, 67-110, and 130-260 microM, respectively. It is concluded that the pharmacological properties of FAAH in the different subcellular fractions are very similar.

Serotonin stimulation of calcium mobilisation in human platelets: choice of units of measurement, effects of age and tobacco use, and correlation with serotonin2A receptor density.

The calcium responses to serotonin and thrombin, as assessed using the fluorescent indicator Fura-2, have been investigated in platelets taken from 59 non-smokers and 17 smokers. The peak responses above baseline, calculated either as fluorescence ratios or calibrated calcium concentrations, were in the order of magnitude thrombin 520 mU/ml > thrombin 52 mU/ml >> serotonin 1 micromol/l approximately serotonin 100 micromol/l. Multiple regression analyses indicated that the responses to 1 micromol/l serotonin, but not the responses to thrombin, were significantly correlated with the serotonin2A receptor density measured using [3H]LSD as radioligand. No effects of age, gender, smoking habit or the time of year of sampling were seen on the calcium responses to serotonin and thrombin. It is concluded that cellular processes distal to the serotonin2A receptor recognition site may compensate to some extent for the large differences in recognition site expression, thus underlining the importance of providing a functional correlate in addition to [3H]LSD binding site densities when studying platelet serotonin2A receptors in neuropsychiatric disorders. The measurement of Ca2+ responses to serotonin provides a useful such functional correlate.

Inhibition of anandamide hydrolysis by the enantiomers of ibuprofen, ketorolac, and flurbiprofen.

The endogenous cannabimimetic anandamide is hydrolyzed by a fatty acid amide hydrolase to yield arachidonic acid and ethanolamine. In the present study, the regional distribution of the activity and its sensitivity to inhibition by the enantiomers of ibuprofen, ketorolac, and flurbiprofen has been investigated. The rate of [3H]anandamide hydrolysis was found in both 7-week-old and 90-week-old rats to be in the order hippocampus > cerebral cortex > cerebellum > striatum approximately midbrain, with higher rates of hydrolysis for the 7-week-old rats than for the 90-week-old rats. In whole brain (minus cerebellum), the R(-)-enantiomer of ibuprofen was a mixed-type inhibitor of anandamide hydrolysis and was approximately 2-3 times more potent than the S(+)-enantiomer, IC50 values of 230 and 750 microM, respectively, being found. A similar pattern of inhibition of anandamide hydrolysis was seen when intact C6 rat glioma cells were used. Ketorolac inhibited rat brain anandamide hydrolysis, with IC50 values of 50, 440, and 80 microM being found for the R-, S-, and R,S-forms, respectively. The IC50 value for R-flurbiprofen (60 microM) was similar to the IC50 value for the S-enantiomer (50 microM). These data demonstrate that there is no dramatic enantiomeric selectivity of NSAID compounds as inhibitors of fatty acid amide hydrolase enzyme(s) responsible for the hydrolysis of anandamide. The enantiomers of flurbiprofen and R-ketorolac are the most potent NSAID inhibitors of fatty acid amide hydrolase yet reported.

Characterization of the enzymatic activity for biphasic competition by guanoxabenz (1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine) at alpha2-adrenoceptors. I. Description of an enzymatic activity in spleen membranes.

The mechanism for formation of high-affinity binding of 1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine (guanoxabenz) to alpha2-adrenoceptors was studied in particulate fractions from the rat spleen. The proportion of apparent high versus low-affinity alpha2-adrenoceptor binding sites increased with increasing incubation time and was also augmented by Mg2+ ions. The formation of high-affinity guanoxabenz binding seemed to be inhibited by a series of N-hydroxyguanidine analogs to guanoxabenz, as well as by a series of metabolic inhibitors that included allopurinol, 1-chloro-2,4-dinitrobenzene, 5,5'-dithiobis-(2-nitrobenzoic acid), cibacron blue, phenyl-p-benzoquinone, didox, and trimidox. The formation of guanoxabenz high-affinity binding was also inhibited in a time- and concentration-dependent fashion by preincubating the membranes with the LW03 N-hydroxyguanidine analogue of guanoxabenz. Moreover, when the spleen membranes were extensively washed for 30 min with buffers at 25 degrees, the guanoxabenz high-affinity binding disappeared. However, when these washed membranes were supplemented with xanthine, the apparent affinity of guanoxabenz increased four to five-fold. Taken together, all data were compatible with the theory that the formation of high-affinity binding was dependent on the generation of a guanoxabenz metabolite that showed an approximate 100-fold greater affinity for the alpha2-adrenoceptors than guanoxabenz itself. Because the most potent blocker of the formation of high-affinity binding was allopurinol (apart from some N-hydroxyguanidine analogs to guanoxabenz) and since the activity could be restored with xanthine, a likely candidate responsible for the metabolic activation is xanthine oxidase.

Serotonin-stimulated calcium responses in human platelets: assay buffer dependency.

In Fura-2 labelled human platelets, the observed calcium response to serotonin stimulation is increased if the assay is conducted in a Krebs buffer compared with the HEPES buffer system usually used. The serotonin response in the Krebs buffer system was concentration dependent with an EC50 value of approximately 0.3 microM and was blocked by nanomolar concentrations of the serotonin2-receptor antagonist methiothepin. In a series of samples collected from 11 persons, a reasonable correlation was seen between the response to 1 microM serotonin and the serotonin2A-receptor density measured in corresponding membrane preparations. The calcium response to serotonin was reduced by treatment with hydrogen peroxide. It is concluded that the use of the Krebs buffer gives a more sensitive response of the Fura-2 loaded human platelets to serotonin stimulation than does the use of HEPES buffer and that the increased sensitivity is achieved without affecting the pharmacological properties of the response. For the response to thrombin, the response intensity is affected by the pH of the buffer used but is not sensitive to the buffer composition.

Comparison of the effects of hydrogen peroxide, 4-hydroxy-2-nonenal and beta-amyloid (25-35) upon calcium signalling.

The neurotoxic beta-amyloid (Abeta) peptide fragment Abeta(25-35) has been suggested to exert its deleterious effects on cells via production of hydrogen peroxide. In human platelets and in the presence of DMSO to prevent production of hydroxyl radicals from hydrogen peroxide, both Abeta(25-35) and hydrogen peroxide were found to increase intracellular calcium levels. Hydrogen peroxide in addition reduced the calcium response to thrombin, whereas this was not seen with Abeta(25-35). A similar pattern of effects to those seen with hydrogen peroxide were also seen with the neurotoxic aldehyde lipid peroxidation product 4-hydroxy-2-nonenal (HNE). The initial increase in calcium produced by hydrogen peroxide was not affected by EGTA, but was partially prevented by dithiothreitol. The calcium response to Abeta(25-35) [which was also seen with Abeta(1-40) and Abeta(1-42) but not with the inactive peptide Abeta(40-1)] consisted of an EGTA-sensitive and an EGTA-resistant component, of which the latter was also sensitive to DTT. Hydrogen peroxide increased basal phosphoinositide breakdown in rat brain miniprisms and decreased the responses to noradrenaline, carbachol and veratrine. The specific binding of [3H]inositol-1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) to its receptor recognition site in human platelet membranes was increased by Abeta(25-35) but remained unchanged following hydrogen peroxide treatment. It is concluded that under conditions where production of hydroxyl radicals from hydrogen peroxide is blocked, hydrogen peroxide and Abeta(25-35) produce their effects on calcium by affecting the mobilisation of intracellular calcium. The qualitative differences in the calcium responses of these two agents can be explained (a) by an additional effect of Abeta(25-35) upon calcium entry and (b) by differences in their effects upon the Ins(1,4,5)P3 receptor.

The sulphydryl oxidizing reagent diamide affects phosphoinositide-mediated signal transduction: implications for the pathogenesis of Alzheimer's disease.

In fura-2-labelled human platelets, the thiol oxidising agent diamide decreases the intracellular calcium response to thrombin and serotonin without affecting the basal calcium levels. The effect of diamide on the thrombin response could be prevented by pre-treatment with dithiothreitol (DTT) and reduced when DTT was added 60 s after diamide. The effects of diamide and hydrogen peroxide on the thrombin response were additive. Hydrogen peroxide also produced a calcium response per se, but this response was not affected by diamide. Hydrogen peroxide increased rat brain phosphoinositide hydrolysis and reduced the response to carbachol and noradrenaline, whereas diamide was without effect. The binding of [3H]inositol-1,4,5-trisphosphate to human platelet membranes was inhibited by diamide but not by hydrogen peroxide. Thus diamide affects the phosphoinositide signal transduction pathway in a qualitatively different manner from that found with hydrogen peroxide. It is suggested that oxidative stress may contribute to the disturbances in the phosphoinositide transduction pathway that are found in Alzheimer's disease.

Propofol inhibits veratrine-stimulated phosphoinositide breakdown in the rat brain.

The intravenous general anesthetic propofol was found to inhibit the phosphoinositide breakdown response to veratrine with an IC50 value of 79 microM. This inhibition was not affected by the addition of flurazepam (0.1, 1, 10 microM) to the assay. The responses to noradrenaline and carbachol were not affected by propofol. It is concluded that inhibition of veratrine-stimulated phosphoinositide breakdown can be used as a simple model system to investigate the potencies at sodium channels of general and as well as local anesthetics.

Ibuprofen inhibits rat brain deamidation of anandamide at pharmacologically relevant concentrations. Mode of inhibition and structure-activity relationship.

The ability of rat brain (minus cerebellum) homogenates to deamidate arachidonyl ethanolamide (anandamide) was determined with a custom-synthesized substrate, arachidonyl ethanolamide-[1-3H] ([3H]anandamide). Conditions whereby initial velocities were measured were established. The homogenates deamidated anandamide with a Km value of 0.8 microM and a Vmax value of 1.73 nmol . (mg protein)-1 . min-1. The deamidation of 2 microM -3H-anandamide was inhibited by phenylmethylsulfonyl fluoride and arachidonyl trifluoromethyl ketone with IC50 values of 3.7 and 0.23 microM, respectively. Ibuprofen inhibited anandamide deamidation in a mixed fashion, with Ki and K'i values of 82 and 1420 microM. At an anandamide concentration of 2 microM, the IC50 values (in microM) of a series of compounds related in structure to ibuprofen were as follows: suprofen, 170; ibuprofen, 270; fenoprofen, 480; naproxen, 550; ketoprofen, 650; diclofenac, approximately 1000. Sulindac produced 27% inhibition at a concentration of 1000 microM, whereas isobutyric acid, hydrocinnamic acid, acetylsalicylic acid and acetaminophen were essentially inactive at concentrations

Quantification of alpha2A and alpha2C adrenoceptors in the rat striatum and in different regions of the spinal cord.

The alpha2C-adrenoceptor preferring radioligand [3H]-MK912 was used for labelling alpha2A- and alpha2C-adrenoceptors in the rat striatum, in the cervical, thoracic and lumbar parts of the spinal cord, and in the dorsal and ventral halves of the spinal cord. In addition, guanfacine was used as a tool to delineate the alpha2A- and alpha2C-adrenoceptors. In the striatum the sites were 72% alpha2A- and 28% alpha2C-adrenoceptors, while in all regions of the spinal cord the proportions of the sites were about 96% alpha2A- and 4% alpha2C-adrenoceptors. A multi-curve experimental design and computer analysis was used in order to enable the accurate quantification of the alpha2A- and alpha2C-adrenoceptors in the striatum and spinal cord.

Comparison of the effects of a series of kappa-opioid receptor agonists upon sodium channel function in rat brain miniprisms.

The blockade of veratrine-stimulated phosphoinositide breakdown in rat cerebral cortical miniprisms has been used as a model of drug action on voltage-dependent sodium channels. The kappa-opioid agonists bremazocine, (+/-)- and (+)-trans-U-50488, U-62066 (spiradoline) and U-69593 inhibited the response to veratrine with IC50 values of 35, 13, 15, 9, and > 100 microM, respectively. Bremazocine, at concentrations inhibiting the response to veratrine, did not inhibit the phosphoinositide breakdown response to the sodium ionophore monensin, indicating the specificity of the assay for sodium channels. The inhibitory actions of bremazocine upon veratrine-stimulated phosphoinositide breakdown were not antagonised by naloxone. This study thus confirms previous data suggesting that the kappa-opioid receptor agonists can affect Na(+)-channel function in a manner unrelated to their actions at kappa-opioid receptors. However, for the compounds tested, such effects are only found at rather high concentrations of the compounds.

Ibuprofen inhibits the metabolism of the endogenous cannabimimetic agent anandamide.

A measure of the metabolism of anandamide, an endogenous cannabimimetic agent, by rat cerebellar membrane preparations was obtained by following the time-dependent reduction in potency of this compound towards inhibition of binding of the high-affinity cannabinoid agonist ligand [3H]WIN 55212-2 to cannabinoid receptors. Thus for example, incubation of the membranes with 100 nM anandamide for 0, 10 and 30 min. prior to addition of [3H]WIN 55212-2 and phenylmethylsulphonyl fluoride (to inhibit the activity of anandamide amidase, thereby blocking further anandamide metabolism during the binding assay) produced 57 +/- 3, 38 +/- 5 and 19 +/- 7% inhibition, respectively, of [3H]WIN 55212-2 binding. This time-dependent effect was blocked by ibuprofen but not by acetyl salicylic acid, sulindac, acetaminophen or to any significant extent by ketoprofen and naproxen. Preliminary experiments using a direct assay of anandamide amidase with [14C]anandamide as ligand gave an IC50 value for ibuprofen of approximately 400 microM. The potency of ibuprofen as an inhibitor of anandamide metabolism was of the same order of magnitude as required for inhibition of cyclooxygenase-2 in cell-free systems and of the peak plasma concentrations of this drug following a 2 x 200 mg dose regimen. It is concluded that following therapeutic doses of ibuprofen, the metabolism of anandamide may be affected.