Rat Spleen Membranes Research Articles

Identification of Novel Endogenous Cytochrome P450 Arachidonate Metabolites with High Affinity for Cannabinoid Receptors

Arachidonic acid is an essential constituent of cell membranes that is esterified to the sn-2-position of glycerophospholipids and is released from selected lipid pools by phospholipase cleavage. The released arachidonic acid can be metabolized by three enzymatic pathways: the cyclooxygenase pathway forming prostaglandins and thromboxanes, the lipoxygenase pathway generating leukotrienes and lipoxins, and the cytochrome P450 (cP450) pathway producing epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids. The present study describes a novel group of cP450 epoxygenase-dependent metabolites of arachidonic acid, termed 2-epoxyeicosatrienoylglycerols (2-EG), including two regioisomers, 2-(11,12-epoxyeicosatrienoyl)glycerol (2-11,12-EG) and 2-(14,15-epoxyeicosatrienoyl)glycerol (2-14,15-EG), which are both produced in the kidney and spleen, whereas 2-11,12-EG is also detected in the brain. Both 2-11,12-EG and 2-14,15-EG activated the two cannabinoid (CB) receptor subtypes, CB1 and CB2, with high affinity and elicited biological responses in cultured cells expressing CB receptors and in intact animals. In contrast, the parental arachidonic acid and epoxyeicosatrienoic acids failed to activate CB1 or CB2 receptors. Thus, these cP450 epoxygenase-dependent metabolites are a novel class of endogenously produced, biologically active lipid mediators with the characteristics of endocannabinoids. This is the first evidence of a cytochrome P450-dependent arachidonate metabolite that can activate G-protein-coupled cell membrane receptors and suggests a functional link between the cytochrome P450 enzyme system and the endocannabinoid system.

Pyrazole derivatives as partial agonists for the nicotinic acid receptor.

Nicotinic acid as a hypolipidemic agent appears unique due to its potential to increase HDL cholesterol levels to a greater extent than other drugs. However, it has some side effects, among which severe skin flushing is the most frequent and often limits patients' compliance. In a search for novel agonists for the recently identified and cloned G protein-coupled nicotinic acid receptor, we synthesized a series of substituted pyrazole-3-carboxylic acids that proved to have substantial affinity for this receptor. The affinities were measured by inhibition of [(3)H]nicotinic acid binding to rat spleen membranes. Potencies and intrinsic activities relative to nicotinic acid were determined by their effects on [(35)S]GTPgammaS binding to rat adipocyte and spleen membranes. Interestingly, most compounds were partial agonists. In particular, 2-diazabicyclo[3,3,0(4,8)]octa-3,8-diene-3-carboxylic acid (4c) and 5-propylpyrazole-3-carboxylic acid (4f) proved active with K(i) values of approximately 0.15 microM and EC(50) values of approximately 6 microM, while their intrinsic activity was only approximately 50% when compared to nicotinic acid. Even slightly more active was 5-butylpyrazole-3-carboxylic acid (4g) with a K(i) value of 0.072 microM, an EC(50) value of 4.12 microM, and a relative intrinsic activity of 75%. Of the aralkyl derivatives, 4q (5-(3-chlorobenzyl)pyrazole-3-carboxylic acid) was the most active with a relatively low intrinsic activity of 39%. Partial agonism of the pyrazole derivatives was confirmed by inhibition of G protein activation in response to nicotinic acid by these compounds. The pyrazoles both inhibited the maximum effect elicited by 100 microM nicotinic acid and concentration dependently shifted nicotinic acid concentration-response curves to the right, pointing to a competitive mechanism of action.

N-acyl-dopamines: novel synthetic CB1 cannabinoid-receptor ligands and inhibitors of anandamide inactivation with cannabimimetic activity in vitro and in vivo

We reported previously that synthetic amides of polyunsaturated fatty acids with bioactive amines can result in substances that interact with proteins of the endogenous cannabinoid system (ECS). Here we synthesized a series of N-acyl-dopamines (NADAs) and studied their effects on the anandamide membrane transporter, the anandamide amidohydrolase (fatty acid amide hydrolase, FAAH) and the two cannabinoid receptor subtypes, CB(1) and CB(2). NADAs competitively inhibited FAAH from N18TG2 cells (IC(50)=19-100 microM), as well as the binding of the selective CB(1) receptor ligand, [(3)H]SR141716A, to rat brain membranes (K(i)=250-3900 nM). The arachidonoyl (20:4 omega 6), eicosapentaenoyl (20:5 omega 3), docosapentaenoyl (22:5 omega 3), alpha-linolenoyl (18:3 omega 3) and pinolenoyl (5c,9c,12c 18:3 omega 6) homologues were also found to inhibit the anandamide membrane transporter in RBL-2H3 basophilic leukaemia and C6 glioma cells (IC(50)=17.5-33 microM). NADAs did not inhibit the binding of the CB(1)/CB(2) receptor ligand, [(3)H]WIN55,212-2, to rat spleen membranes (K(i)>10 microM). N-arachidonyl-dopamine (AA-DA) exhibited 40-fold selectivity for CB(1) (K(i)=250 nM) over CB(2) receptors, and N-docosapentaenoyl-dopamine exhibited 4-fold selectivity for the anandamide transporter over FAAH. AA-DA (0.1-10 microM) did not displace D1 and D2 dopamine-receptor high-affinity ligands from rat brain membranes, thus suggesting that this compound has little affinity for these receptors. AA-DA was more potent and efficacious than anandamide as a CB(1) agonist, as assessed by measuring the stimulatory effect on intracellular Ca(2+) mobilization in undifferentiated N18TG2 neuroblastoma cells. This effect of AA-DA was counteracted by the CB(1) antagonist SR141716A. AA-DA behaved as a CB(1) agonist in vivo by inducing hypothermia, hypo-locomotion, catalepsy and analgesia in mice (1-10 mg/kg). Finally, AA-DA potently inhibited (IC(50)=0.25 microM) the proliferation of human breast MCF-7 cancer cells, thus behaving like other CB(1) agonists. Also this effect was counteracted by SR141716A but not by the D2 antagonist haloperidol. We conclude that NADAs, and AA-DA in particular, may be novel and useful probes for the study of the ECS.

Characterization of chemokine CCR3 agonist-mediated eosinophil recruitment in the Brown-Norway rat.

1. The ability of various C-C chemokines to elicit tissue eosinophil infiltration following intradermal injection or peripheral blood eosinophilia following intravenous injection were compared in the Brown-Norway rat. 2. Eotaxin (0.1 - 3 microg site-1) of human and murine origin produced equivalent, dose-dependent increases in eosinophil peroxidase activity in rat dermis 4 h post-injection. 3. Human eotaxin-2 was equipotent with human eotaxin in terms of dermal eosinophil recruitment. Other human CCR3 agonists, such as MCP-3, RANTES and MCP-4 failed to increase dermal eosinophil peroxidase activity at doses up to 1 microg site-1 whereas the latter did produce a small effect at 3 microg site-1. 4. Consistent with observations in vivo, human eotaxin displaced [125I]-eotaxin from rat spleen membranes more potently (IC50=2 nM) than did MCP-4 (IC50=500 nM). RANTES did not compete with the radiolabelled chemokine at concentrations up to 1 microM. 5. Human eotaxin (5 microg) administered intravenously increased circulating eosinophils approximately 3 fold whereas MCP-4 (5 microg i.v.) increased circulating monocytes approximately 3 fold without affecting eosinophil numbers. 6. Dexamethasone pretreatment inhibited eotaxin-induced dermal eosinophil influx only at a steroid dose (0.1 mg kg-1, s.c.) which significantly reduced circulating eosinophil numbers. The steroid also reduced eosinophilia in peripheral blood resulting from systemic eotaxin administration (5 microg, i.v.). 7. These data suggest differences in rat CCR3 relative to other species as surmised from a distinctive rank order of chemokine potency. In addition to its chemotactic effects eotaxin, but not MCP-4, promotes eosinophil recruitment into the circulation. One of the mechanisms by which glucocorticoids, such as dexamethasone, acutely inhibits eotaxin-induced dermal eosinophil influx is to diminish the circulating numbers of these cells available for tissue recruitment.

Allosteric modulation of peripheral sigma binding sites by a new selective ligand: SR 31747

The interactions of a new compound SR 31747 with sigma sites were examined in rat spleen membranes and in human peripheral blood leukocytes (PBL). Nanomolar concentrations of SR 31747 selectively inhibited in a non-competitive manner the binding of the prototypic sigma ligands [3H](+)-pentazocine, [3H](+)-3PPP and [3H]DTG on rat spleen membranes. Characterization of SR 31747 binding sites using [3H]SR 31747 as a ligand showed that this compound binds reversibly, with high affinity to one class of sites on rat spleen membranes (Kd 0.66 nM, Bmax 5646 fmol/mg protein). The pharmacological profile of [3H]SR 31747 binding sites was consistent with the presence of specific sites distinct from classical sigma 1 and sigma 2 receptor subtypes strongly suggesting an allosteric modulation of sigma sites by SR 31747. Similarly, [3H]SR 31747 binding sites were demonstrated on human PBL and also on purified subpopulations of human mononuclear cells (granulocytes, NK cells, T4, T8 and B lymphocytes). Administered to mice by i.p. or oral route 30 min before sacrifice, SR 31747 strongly inhibited the binding of [3H](+)-3PPP to mice spleen membranes with ED50 values of 0.18 and 1.43 mg/kg, respectively. Taken together these results could suggest a potential immunological activity of SR 31747 either directly or through allosteric modulation of peripheral sigma sites.

Determination of alpha 1-adrenoceptor subtype selectivity by [3H]-prazosin displacement studies in guinea-pig cerebral cortex and rat spleen membranes.

1. [3H]-prazosin homogeneously labels alpha 1-adrenoceptors in guinea-pig cerebral cortex and rat spleen membranes with dissociation constants of 1.28 and 1.49 x 10(-10) M respectively. 2. Phentolamine and WB 4101 displacement studies show that guinea-pig cerebral cortex contains 30% alpha 1A- and 70% alpha 1B-adrenoceptor subtypes, whereas rat spleen contains a virtually homogeneous alpha 1B-adrenoceptor subtype population. The alpha 1-adrenoceptor population of rat thoracic aorta is predominantly of the alpha 1A-adrenoceptor subtype, and in guinea-pig thoracic aorta it is mainly of the alpha 1B-adrenoceptor subtype. 3. Half of the compounds displacing [3H]-prazosin bound to guinea-pig cerebral cortex membranes display alpha 1A-adrenoceptor selectivity. Among these compounds, WB 4101 and methoxamine are most selective, displaying selectivity ratios of approximately 38 and approximately 26 respectively. 4. The affinity constants of the non-selective compounds for the alpha 1-adrenoceptor in guinea-pig cerebral cortex membranes correlate well with the affinity constants obtained for alpha 1B-adrenoceptors in rat spleen membranes. The affinities of selective compounds for the alpha 1B-adrenoceptor subtype in guinea-pig cerebral cortex correlate very well with their affinity for alpha 1B-adrenoceptor in the rat spleen homogenate. Both regression lines coincide with the line of identity. The affinity constants of selective compounds for the alpha 1A-adrenoceptors in guinea-pig cerebral cortex only apparently correlate with the affinity for either the alpha 1B-adrenoceptors in guinea-pig cerebral cortex or in the rat spleen. Regression analyses indicate a straight line relationship (r2>0.9) between pKEA and Pk1B but the regression lines deviate from the line of identity.

Comparison of .ALPHA.1-Adrenoceptors between Rat Brain and Spleen.

Scatchard analyses of 3H-prazosin binding in rat brain membranes showed biphasic curves, which identified the presence of alpha 1High- and alpha 1Low-affinity sites. The alpha 1High-affinity site was completely inhibited by 0.1 microM phenoxybenzamine. On the other hand, 3H-prazosin binding in rat spleen membranes resulted in linear curves that were identical to the binding curve for the alpha 1High-affinity site in the brain. The displacement potencies of alpha 1-adrenergic antagonists were characterized by 3H-prazosin binding to alpha 1High-affinity sites in the rat spleen and brain and alpha 1Low-affinity sites in the brain in the presence of 0.1 microM of phenoxybenzamine. The affinities of WB-4101, phenoxybenzamine, phentolamine, chlorpromazine, labetalol and nifedipine for brain alpha 1High-affinity sites were significantly higher than those in the spleen. The affinities of most ligands for alpha 1Low-affinity sites were significantly lower than those for both alpha 1High-affinity sites in the brain and spleen, but chlorethylclonidine was significantly selective for alpha 1Low-affinity sites, and bunazosin, dibenamine and 5HT had the same affinities for the alpha 1Low- and both alpha 1High-affinity sites. These results show that two alpha 1-adrenoceptor subtypes, alpha 1High- and alpha 1Low-affinity, are present in the rat brain and that a different alpha 1High-subtype, exists in the rat spleen.

51] Purification and molecular cloning of Rat Ia antigens

This chapter describes the purification of Ia glycoproteins from detergent solubilized rat spleen membranes using monoclonal antibody affinity chromatography and the characterization of the purified Ia molecules. Methods are also described for the purification of mRNA from rat spleen and the molecular cloning of cDNA coding for rat Ia-A antigens. The chapter reviews that the Ia antigens are a set of highly polymorphic cell surface molecules involved in many cell-cell interactions and coded for by the major histocompatibility complex (MHC). The polymorphism of the Ia antigens is directly related to their function, as these molecules are thought to be the products of the immune response genes which genetically determine the outcome of many types of immune responses. These have been identified in several different mammalian species and are composed of two noncovalently bonded glycosylated polypeptides referred to as the α and β chains. The chapter also discusses that the Ia glycoproteins show a restricted tissue distribution being present on B lymphocytes, dendritic cells, some macrophages and thymocytes, and on epithelial and reticular cells of thymus and spleen. These molecules are not generally found on the majority of T lymphocytes or other cells. The rat MHC, called the RT1 complex, codes for two sets of Ia antigens referred to as Ia-A and Ia-E which correlate to the mouse H-2 I-A and I-E Ia antigens.

Pharmacological characteristics of beta-adrenoceptor binding sites in intact and sympathectomized rat spleen

[3H]-(--)-Dihydroalprenolol ([3H]-DHA) binds to rat spleen membranes with a dissociation equilibrium constant (KD) of about 0.7 nM and a maximal number of binding sites of 272 fmoles x mg protein-1. Approximately 90% of the sites labelled by 1 nM [3H]-DHA possess classical properties of beta-adrenoceptors. The labelled ligand is stereospecifically displaced by the isomers of propranolol and the order of potency of agonists is: isoprenaline greater than adrenaline greater than noradrenaline. Highly selective beta 1 antagonists such as practolol and atenolol displaced [3H]-DHA from spleen membranes in a biphasic manner indicating the co-existence of beta 1 and beta 2 sites (30--35% beta 1; 65--70% beta 2) in this tissue. Support for this classification was provided by the binding of the beta 2 agonist procaterol to spleen membranes. This drug possessed high affinity only for those sites that have low affinity for the beta 1 selective agents. Chemical sympathectomy induced by chronic 6-hydroxydopamine administration did not alter the number or the pharmacological properties of beta-adrenoceptor binding sites. The results suggest that both beta 1 and beta 2 adrenoceptors are probably postsynaptic in spleen.