Monoclonal anti-idiotypic antibodies to opioid receptors

Two monoclonal anti-idiotypic antibodies (anti-Id-135 and anti-Id-14, both of the IgM class) which interact with the binding site of opioid receptors were generated. A monoclonal anti-beta-endorphin antibody (3-E7) which displays binding characteristics for opioid ligands similar to opioid receptors served as the antigen (Gramsch, C., Meo, T., Riethmüller, G., and Herz, A., (1983) J. Neurochem. 40, 1220-1226; Meo, T., Gramsch, C., Inan, R., H?llt, V., Weber, E., Herz, A., and Riethmüller, G. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4048-4088) and the hybridomas obtained were screened for anti-idiotypic antibodies with Fab fragments of 3-E7. The anti-idiotypes were then screened for opioid binding to rat brain membrane receptors, yielding several positive clones two of which were more intensively studied. Both anti-idiotypic antibodies were about equally potent in displacing the mu- and delta-opioid receptor ligands [3H]dihydromorphine, 125I-labeled beta-endorphin, [D-Ala2, D-Leu5-3H]enkephalin and [3H]naloxone from rat brain membrane opioid receptors; no interaction was observed with the kappa-ligands [3H]ethylketazocine or [3H]bremazocine. The anti-idiotypic antibodies were able to precipitate [3H] diprenorphine binding sites from solubilized opioid receptor preparations. In addition, both antibodies showed opioid antagonistic properties as demonstrated by their abilities to block the inhibitory effect of [D-Ala2, D-Leu5-3H]enkephalin on prostaglandin E1-stimulated cAMP accumulation in NG 108-15 hybrid cells. Our findings demonstrate the successful generation of monoclonal antibodies interacting with membrane-bound and solubilized opioid receptors of the mu- and delta-type.     

The specificity of the binding of platelet activating factor (PAF) to anti-PAF antibodies.

Quantitative hapten inhibition experiments employing sheep anti-PAF antibodies and selected PAF analogues were undertaken with the aim of defining the antigenic determinant structures complementary to the antibody combining sites. The most important fine structural features for inhibition of antibody to PAF were shown to be an acetyl group at position 2 of the phospholipid glycerol backbone and an ether group at position 1. Of the naturally occurring compounds, C16- and C18:1-PAF proved to be the most potent inhibitors and more active than C18-PAF while phospholipids with a propionyl, butyryl or hexanoyl group at position 2 showed either weak or no inhibitory activity. The 1-acyl, thioether and deoxy analogues proved inactive. Variations in the polar head group of PAF were found to be less critical with, for example, the dimethyl and ethanolamine derivatives retaining some activity. This antibody recognition pattern is very similar to that of the PAF receptor, although the antibodies appear to have a more specific requirement for an acyl linkage at position 2.     

Synthesis of thioanalogs of platelet activating factor (PAF)

Synthesis of thioalkyl, thioacetyl and phosphothionyl PAF analogues was carried out starting with corresponding monoalkyl glycerol ethers. The synthetic route was based on preparation of racemic phosphatidylcholines and subsequent hydrolysis with phospholipase A2 to afford isomers having natural configuration.     

A bioregulator of lipid nature--a platelet activating factor (PAF)

The review concerns metabolism, immunological and antihypertensive action of platelet activating factor (PAF), a bioregulator of lipid nature. Major synthetic approaches to PAF and its analogues are described. The effects of structural modification on the physiological activity of PAF are considered.     

Tumor growth, angiogenesis and inflammation in mice lacking receptors for platelet activating factor (PAF)

Tumor growth is associated with angiogenesis and inflammation and the endogenous lipid, platelet activating factor (PAF), is a pro-inflammatory and pro-angiogenic mediator. We therefore measured tumor growth, angiogenesis and inflammation in normal (WT) mice and those lacking the receptor for PAF, through gene deletion (PAFR-KO). Growth of solid tumors derived from colon 26 cells was not altered but that from Ehrlich cells was markedly (5-fold) increased in the PAFR-KO mice, relative to the WT strain. Angiogenesis, as tumor content of VEGF or hemoglobin, was increased in both tumors from the mutant strain. Inflammation, as neutrophil and macrophage accumulation and chemokine (CXCL2 and CCL2) content of tumors, was decreased or unchanged in the tumors implying an overall decrease in the inflammatory response in the PAFR-KO strain. We also assessed growth of the Ehrlich tumor in its ascites form, after i.p. injection. Here growth (ascites volume) was inhibited by about 30%, but neutrophil and macrophage numbers were increased in the ascites fluid from the PAFR-KO mice. Angiogenesis in the peritoneal wall, which is not invaded by the tumor cells, was increased but leukocyte infiltration decreased in the mutant strain. Our results show, unexpectedly, that tumor-induced angiogenesis was increased in mice lacking response to PAF, from which we infer that in normal (WT) mice, PAF is anti-angiogenic. Further, although growth was still associated with angiogenesis in PAFR-KO mice, growth was not correlated with inflammation (leukocyte accumulation).     

Pharmacological actions of Y-24180, a new specific antagonist of platelet activating factor (PAF): II. Interactions with PAF and benzodiazepine receptors

The inhibitory effect of Y-24180, 4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H-t hieno [3,2-f][1,2,4]triazolo [4,3-a][1,4]diazepine, on platelet activating factor (PAF)-induced platelet aggregation and the specific binding of 3H-PAF to platelets was compared with other thienodiazepine derivatives, WEB 2086 and etizolam. Y-24180 inhibited PAF-induced rabbit platelet aggregation in vitro (IC50 3.84 nM), but had little effect on adenosine diphosphate- or arachidonic acid-induced aggregation. WEB 2086 and etizolam also showed an inhibitory effect of PAF-induced aggregation (IC50 values are 456 and 6730 nM, respectively). In PAF-induced human platelet aggregation, Y-24180 (IC50 0.84 nM) was more potent than WEB 2086 (IC50 4.21 nM) and etizolam (IC50 998 nM). Y-24180, WEB 2086 and etizolam displaced 3H-PAF binding from the washed-platelets of rabbits with an IC50 value of 3.50, 9.35 and 29.5 nM, respectively. In rabbits, pretreatment with Y-24180 and WEB 2086 antagonized PAF-induced platelet aggregation dose-dependently. The significant inhibitory effect of Y-24180 (1 mg/kg, p.o.) lasted 72 hr after a single dose oral administration. WEB 2086 (10 mg/kg, p.o.) also antagonized the ex vivo response induced by PAF 1 hr after administration, but no significant effect was observed 3 hr after administration. Y-24180 displaced 3H-diazepam binding from the synaptosomal membranes of rat cerebral cortex with a Ki value of 3.68 microM. The affinity of Y-24180 for benzodiazepine(BZP) receptors was lower than those of WEB 2086 and etizolam and was about 1000 times lower than that for PAF receptors in platelets.     

Antibody to platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF)

Specific antibodies to platelet activating factor (PAF) were prepared by immunizing rabbits with a hapten-bovine serum albumin (BSA) conjugate. As the hapten we used the synthetic PAF derivative which is resistant against enzymatic inactivation by plasma or tissues and which can bind to BSA through covalent bonding. Antibody activity was determined by an enzyme-linked immunosorbent assay (ELISA). Anti-PAF IgG reacted strongly with PAF. By means of the ELISA inhibition assay, we found that the antibody did not cross-react with phosphocholine, glycerophosphocholine, dilaurylglycerophosphocholine or PAF analogues which have ethanolamine-type polar head groups instead of choline group.     

Triazolobenzodiazepines competitively inhibit the binding of platelet activating factor (PAF) to human platelets

PAF causes dose dependent platelet aggregation of human platelet rich plasma or gel filtered platelets (GFP). The benzodiazepines alprazolam and triazolam, but not diazepam (1-10 microM), inhibit PAF induced aggregation but have no effect on aggregation induced by other platelet agonists such as ADP, epinephrine and collagen. The IC50 for aggregation by PAF (4 nM) in GFP is 1 microM for both alprazolam and triazolam. The mechanism for this inhibition was explored by studying the binding of 3H-PAF(0.08 nM) to GFP in Tyrodes buffer containing albumin (0.35%), Mg++ (1mM) and Ca++ (0.5mM). GFP was incubated with different doses of the drug for 5 min prior to addition of 3H-PAF. Incubation was then carried out for 60 min at 25 degrees C to achieve binding equilibrium, as previously established. Alprazolam and triazolam, but not diazepam, caused competitive displacement of 3H-PAF from specific binding sites of GFP. The IC50 of alprazolam was 3.8 microM while that of triazolam was 0.82 microM. Lineweaver-Burk plots of 3H-PAF binding in the presence of inhibitor were also consistent with competitive inhibition. These results are consistent with the interpretation that the specific inhibition of PAF induced platelet aggregation by alprazolam and triazolam, respectively, is due to competitive inhibition of binding of PAF to its receptor.     

CV-3988 - a specific antagonist of platelet activating factor (PAF)

CV-3988, rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl 2-thiazolioethyl phosphate was shown to be a specific inhibitor of platelet activating factor (PAF). This compound in concentrations of 3 x 10(-6) to 3 x 10(-5)M inhibited aggregation of rabbit platelets induced by PAF (3 x 10(-8)M), while it had no effect on the aggregation induced by arachidonic acid, ADP, collagen or A-23187. CV-3988 alone even at a concentration of 10(-3)M had no effect on platelet aggregation. The inhibitory action of CV-3988 on the PAF-induced aggregation was independent of the formation of micelles. The PAF (0.1 to 1.0 micrograms/kg, i.v.)-induced hypotension in anesthetized rats was also inhibited dose-dependently by the i.v. administration of CV-3988 (1 and 10 mg/kg), while the hypotensive actions induced by the i.v. administration of acetylcholine (1 micrograms/kg), arachidonic acid (1 mg/kg), bradykinin (10 micrograms/kg), isoproterenol (1 microgram/kg) and histamine (100 micrograms/kg) were not altered by CV-3988 (10 mg/kg, i.v.). All these findings indicate that CV-3988 specifically inhibits the action of PAF in vitro and in vivo. This is the first report of a PAF antagonist which can specifically inhibit the PAF-induced hypotension as well as the PAF-induced platelet aggregation.     

The platelet activating factor (PAF) signaling cascade in systemic inflammatory responses

The platelet-activating factor (PAF) signaling cascade evolved as a component of the repertoire of innate host defenses, but is also an effector pathway in inflammatory and thrombotic diseases. This review focuses on the PAF signaling cascade in systemic inflammatory responses and, specifically, explores its activities in experimental and clinical sepsis and anaphylaxis in the context of the basic biochemistry and biology of signaling via this lipid mediator system.     

Endothelin-induced sudden death and the possible involvement of platelet activating factor (PAF)

Endothelin (5 nmol/kg, i.v.) caused a transient hypotension followed by a lasting hypertension in rats. However, an abrupt fall in the blood pressure was observed in most rats 6 to 30 min after the injection of endothelin and sudden death followed with lethality noted over 60 min. An abnormal electrocardiogram (ECG) (ventricular arrhythmias) was observed in rats injected with endothelin. Endothelin (i.v.) also caused sudden death in mice. Pretreatment (5 or 60 min) with specific PAF antagonists, CV-6209 (0.1-3 mg/kg, i.v.) and WEB 2086 (30 mg/kg, p.o.), and a calcium channel blocker, diltiazem (60 mg/kg, p.o.) prevented death and attenuated the ECG changes induced by endothelin, but CV-6209 did not prevent the blood pressure changes induced by endothelin. CV-6209 (0.5-3 mg/kg, i.v.), WEB 2086, diltiazem and dexamethasone (5 mg/kg, i.v.) protected mice against the death induced by endothelin. On the other hand, aspirin (cyclooxygenase inhibitor, 100 mg/kg, p.o.) did not protect mice from the death. Thus, endothelin is a highly toxic peptide with cardiotoxic effects, and PAF may be involved in the pathogenesis of the sudden death.     

Novel agents combining platelet activating factor (PAF) receptor antagonist with thromboxane synthase inhibitor (TxSI)

Compounds 1 or 2 which possess dual-acting PAF antagonist/TxSI in a previous paper were modified and evaluated for the dual-acting activity. It was found that several compounds were potent dual-acting PAF antagonist/TxSI in and ex vivo. 6-(2-Chlorophenyl)-3-[4-[(E/Z)-6-ethoxycarbonyl-1-(3-pyridyl)-1-hexenyl]phenylmethyl]-8,11-dimethyl-2,3,4,5-tetrahydro-8H-pyrido[4',3': 4,5]thieno[3,2-f]triazolo[4,3-a]diazepine (12) is excellent orally dual-acting PAF antagonist/TxSI.     

Spasmogenic effect of platelet activating factor (PAF) on isolated rat stomach fundus strip

Platelet activating Factor (PAF) produced an increase in resting tension of isolated rat stomach fundus strips. The spasmogenic effect of a 90 nM dose was equivalent to the contraction to 110 nM acetylcholine (ACh). Tissues exposed once to PAF became refractory to re-challenge with a dose of PAF normally producing maximum contraction (desensitization). PAF desensitized tissues remained responsive to the contraction effects of ACh and KCl (80 mM). Lyso-PAF failed to produce any effect. PAF contraction was dose-dependently antagonized by pretreatment of tissues with the PAF receptor antagonist L-652,731. PAF contractions were not blocked by antagonists of cholinergic, adrenergic, histaminergic, and serotonergic receptors, nor by inhibition of cyclooxygenase. PAF is a potent spasmogen on the isolated rat stomach fundus strip, and this effect is PAF and PAF-receptor specific.     

Structure-dependent effects of glucose-containing analogs of platelet activating factor (PAF) on membrane integrity

Synthetic choline-containing phospholipids comprise a new class of compounds with antineoplastic properties. We have investigated the effect of recently synthesized glucose-containing analogs of lysophosphatidylcholine (glyceroglucophospholipid, Glc-PC) and of lysoplatelet activating factor (Glc-PAF) and its C16, C14 and C12 derivatives (ET-16, ET-14, and ET-12) on proliferation of immortalized human keratinocyte (HaCaT) cells. The data were compared to the ability of the compounds to intercalate into phosphatidylserine liposomes and to form lesions in planar bilayer membranes. A correlation between bioactivity and membrane activity was found. The number of molecules that intercalated into phosphatidylserine liposomes depended on the chemical structure of the compounds and was in the order Glc-PAF approximately ET-16 approximately ET-14 > Glc-PC > ET-12. All compounds induced membrane lesions, and the lesion forming activity was in the same order. Similar activity rankings were found for the release of lactate dehydrogenase from HaCaT cells as a measure of lytic activity and for the influence on cell number as a measure of proliferation. In the latter test, however, proliferation was already inhibited at non-toxic concentrations. From these findings, it may be concluded that the intercalation of the compounds at toxic concentrations leads to the formation of membrane lesions and finally results in membrane rupture leading to cell death.     

Activation of guinea pig peritoneal macrophages by platelet activating factor (PAF) and its agonists

The platelet activating factor (PAF: 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) and its analogs were examined to determine their effects on guinea pig peritoneal macrophages. PAF activated macrophages, but its effect on macrophages was much weaker than that observed on platelets: the concentration required for 50% maximum activation was 8.5 X 10(-6) M for macrophages and 2.9 X 10(-10) M for platelets. Three PAF agonists, 1-O-octadecyl-2-O-(N,N-dimethylcarbamoyl)-glycero-3-phosphocholine (Compound I), 1-O-octadecyl-2-acetamido-2-deoxy-glycero-3-phosphocholine (Compound II), and 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine (Compound III), showed higher activity in stimulating macrophage function than PAF. The abilities of these non-metabolizable PAF agonists to activate macrophage paralleled their relative potency to induce platelet activation. The sn-3 enantiomers of PAF and Compound III exhibited activity, while the sn-1 did not. By comparing the activities of derivatives of Compound III, it was shown that the long-chain alkyl-ether group in the glycerol-1 position, a relatively small size of the substituent on the hydroxy group at the sn-2 position, and the choline moiety in the glycerol-3 position must play critical roles in the process of macrophage activation. A specific PAF antagonist, CV3988, which inhibits PAF-induced platelet activation and hypotension, inhibited the activation of macrophages caused by PAF and its agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Approach to dual-acting platelet activating factor (PAF) receptor antagonist/thromboxane synthase inhibitor (TxSI) based on the link of PAF antagonists and TxSIs

A series of compounds (22-36) which possess dual-acting PAF antagonist/TxSI have been generated by the approach of linking the known PAF antagonists and TxSIs, such as Ridogrel (1).     

Syntheses and bioactivities of novel carbamates combining platelet activating factor (PAF) receptor antagonist with thromboxane synthase inhibitor (TxSI)

Synthesis of carbamates 3b which possess dual-acting PAF antagonist/TxSI using unstable esters 1, diazepines 2, K2CO3 and 18-crown-6 is described.     

Alteration of platelet activating factor (PAF) metabolism in rat pulmonary alveolar macrophages and plasma by cigarette smoking.

The pathophysiological role of platelet activating factor (PAF) in smoking-induced disorders was examined in rats exposed daily to smoke for 10, 18 and 26 weeks. The concentration of PAF in bronchoalveolar lavage fluid and the activities of PAF biosynthetic and catabolic enzymes in alveolar macrophages and in plasma were determined. The concentration of PAF in lavage fluid of the smoke-exposed group was significantly lower than that in the sham group for each duration of smoke exposure. The PAF biosynthetic enzyme, acetyl transferase, activity in alveolar macrophages of smoked group was less than that in the sham group although the difference was not statistically significant. PAF catabolic enzyme, acetyl hydrolase, activities in alveolar macrophages and in plasma were all significantly higher in every smoked group than in the sham group. These data indicate that cigarette smoking alters PAF metabolism in the respiratory tract and in plasma and such an alteration may contribute, at least in part, to smoking induced cardiopulmonary disorders.