The specific binding of the platelet-activating factor (PAF) receptor antagonist WEB 2086 and the benzodiazepine flunitrazepam to rat hepatocytes

Thieno-triazolodiazepines WEB 2086 and BN 50739 have been described as the potent PAF receptor antagonists. Binding of radiolabeled [3H]WEB 2086 has been widely employed to characterize PAF receptors in different cells. In a search for a PAF receptor in isolated rat hepatocytes, we discovered that the binding of [3H]WEB to rat hepatocytes was highly specific but had a relatively low affinity with a Kd of 113 nM and Bmax of 0.65 pmol/10(6) cells in freshly isolated cell suspension and Kd of 1.65 muM and Bmax of 2.0 pmol/plate in cultured hepatocytes. No consistent specific binding of [3H]PAF itself was found in the same cell preparations. The binding of [3H]flunitrazepam in the presence of the peripheral type of benzodiazepine receptor antagonist Ro 5-4864 was saturated and exhibited a K(i) of 3.8 nM and Bmax of 3.5 pmol/plate. The central type of benzodiazepine receptor antagonist clonazepam was competed for the [3H]flunitrazepam binding, however with a much lower affinity. Various antagonists inhibited the binding of [3H]WEB 2086 with a rank order BN 50739>Ro 5-4864 > or = clonazepam. Interestingly, bicuculline, specific antagonist of GABA(A) recognition sites, also significantly reduced the binding of [3H]WEB 2086. The binding of [3H]flunitrazepam was inhibited with a rank potency BN 50739>WEB 2086. Taken together, these findings suggest that the specific binding of PAF receptor antagonists WEB 2086 and BN 50739 in rat hepatocytes does not involve PAF receptors and occurs via peripheral benzodiazepine and, possibly GABA(A) receptor sites.     

Administration of a receptor antagonist for platelet-activating factor during equine endotoxaemia

Platelet-activating factor (PAF) is an important mediator of endotoxaemia and various PAF receptor antagonists prevent many of the adverse effects of experimental endotoxaemia in laboratory animals. In this study a specific PAF receptor antagonist was used to investigate the role of PAF in equine endotoxaemia. At an interval of not greater than 10 days, 6 horses were each challenged with endotoxin and endotoxin with concurrent administration of SRI 63-441, a PAF receptor antagonist. The order of the treatments was randomised. Clinical signs, serum biochemical and coagulation profiles, and platelet aggregation in vitro were monitored in all horses for 24 h after treatment. Challenge with endotoxin increased maximal platelet aggregation induced by PAF. This response was blocked by administration of SRI 63-441 concurrently with endotoxin. No changes in percentage maximal platelet aggregation to ADP or collagen were noted after administration of endotoxin. The PAF receptor antagonist delayed the onset of fever, tachycardia, leucopenia and lactic acidaemia. Lack of more profound beneficial alterations of the horses' responses to endotoxin may have been due to the low dose of endotoxin administered in this model or to only partial effectiveness of SRI 63-441 in blocking the effects of endotoxin-induced PAF.     

Molecular mechanisms of transforming growth factor-beta signaling

The present experiments examined the effects of posttraining intrahippocampal injections of the degradative enzyme-resistant methylcarbamyl analog of the bioactive phospholipid platelet-activating factor (mc-PAF) and the platelet-activating factor (PAF) receptor antagonists BN52021 and BN 50730 on memory in male Long-Evans rats trained in a hidden platform version of the Morris water maze. Following an eight-trial training session, rats received a unilateral intrahippocampal injection of mc-PAF (0.5, 1.0, or 2.0 microgram/0.5 microliter), lyso-PAF (1.0 microgram/0.5 microliter), the cell surface PAF receptor antagonist BN 52021 (0.25, 0.5, or 1.0 micrigram/0.5 microliter/, the intracellular PAF receptor antagonist BN 50730 (2.0, 5.0, or 10.0 microgram/0.5 microliter), or vehicle (50% DMSO in 0.9% saline; 0.5 microliter). On a retention test conducted 24 h after training, the escape latencies of rats administered mc-PAF (1.0 or 2.0 microgram) were significantly lower than those of the vehicle-injected controls, demonstrating a memory-enhancing effect of mc-PAF. Injections of lyso-PAF, a structurally similar metabolite of PAF, had no influence on memory, indicating that the memory-enhancing effect of mc-PAF is not caused by membrane perturbation by the phospholipid. The retention test escape latencies of rats administered BN 52021 (0.5 microgram) and BN 50730 (5.0 or 10 microgram) were significantly higher than those of the controls, indicating a memory impairing effect of both PAF antagonists. When mc-PAF, BN 52021, or BN 50730 was administered 2 h posttraining, no effect on retention was observed, indicating a time-dependent effect of the neuroactive substances on memory storage. The findings suggest a role for endogenous PAF in hippocampal-dependent memory processes.     

Triflavin potentiates the antiplatelet activity of platelet activating factor receptor antagonist on activated neutrophil-induced platelet aggregation

In this study, specific platelet activating factor (PAF) receptor antagonist ginkgolide B (BN52021) was tested for its antiplatelet activity in zymosan activated polymorphonuclear neutrophil-induced platelet aggregation. Triflavin was also tested for its antiplatelet activity compared with PAF receptor antagonist. Triflavin, an Arg-Gly-Asp-containing disintegrin purified from venom peptide inhibited platelet aggregation by interfering with the interaction of fibrinogen with the glycoprotein IIb/IIIa complex. Furthermore, we also report an efficient high resolution method for quantitative analysis of PAF using high-performance capillary electrophoresis (HPCE). The supernatant of polymorphonuclear neutrophils after their activation by opsonized zymosan induces the aggregation of washed rabbit platelets. In rabbit platelets, BN52021 (100-1000 microM) only partially inhibited activated polymorphonuclear neutrophil-induced platelet aggregation, and its maximal inhibition was estimated to be about 79%. Triflavin also partially inhibited platelet aggregation about 82% induced by activated polymorphonuclear neutrophils. Furthermore, after treatment with a combination of triflavin (0.26 microM) with various concentrations of BN52021 (4-1000 microM), the inhibitory effect of platelet aggregation was almost completely. This inhibition was greater than that produced by the individual drugs alone. These results indicate that a combination of glycoprotein IIb/IIIa complex and PAF receptor antagonist could completely inhibit activated polymorphonuclear neutrophil-induced platelet aggregation. In addition, the amount of PAF released from zymosan (6 mg/ml)-activated polymorphonuclear neutrophils was accurately calculated about 11.8+/-1.5 ng/10(6) cells, and did not further increase even at a high concentration of zymosan (10 mg/ml). These results suggest that PAF play a major role in the interaction between platelets and polymorphonuclear neutrophils. This interaction may be important in the pathogenesis of thrombosis and inflammatory diseases. Our present findings support the hypothesis that combination therapy with glycoprotein IIb/IIIa complex antagonists and PAF receptor antagonists may represent a new approach to the treatment of ischemic disorders.     

The platelet-activating factor antagonist BN 52021 attenuates hypoxic-ischemic brain injury in the immature rat

Platelet-activating factor (PAF) is overproduced in ischemic brain. Although postischemic PAF antagonist administration protects the mature brain in some models, little is known about the effects of PAF antagonists in the immature brain. We hypothesized that the PAF antagonist BN 52021 would attenuate perinatal cerebral hypoxic-ischemic injury. To elicit focal hypoxic-ischemic brain injury, 7-d-old (P7) rats (n = 111) underwent right carotid ligation, followed by 2.5-3.25 h of hypoxia (fractional concentration of inspired O2 = 0.08). BN 52021 neuroprotection was evaluated in three groups of experiments: 1) 25 mg/kg/dose, 0 and 2 h posthypoxia; 2), 25 mg/kg/dose immediately before and 1 h after hypoxia; and 3) posthypoxia-ischemia treatment with BN 52021 12.5, 25, or 50 mg/kg/dose in 2 doses 0 and 2 h after hypoxia. All experiments included concurrent vehicle-injected controls. To quantitate severity of injury, bilateral regional cross-sectional areas (groups 1 and 2) or hemisphere weights (group 3) were evaluated on P12. Both pre- and posthypoxic treatment with BN 52021 (25 mg/kg/dose, two serial doses) decreased the incidence of cerebral infarction from 90% to about 30% (p < 0.02, Fisher's exact test). Measurement of cross-sectional areas confirmed neuroprotection and indicated some benefit of pre- over posthypoxic-ischemic treatment in hippocampus and cortex. Over the dose range tested, the neuroprotective effect of BN 52021 administration was not dose-dependent. In contrast, BN 52021 did not attenuate N-methyl-D-aspartate-induced hippocampal excitotoxic injury in P7 rats. Either prophylactic or "rescue" administration of PAF antagonists decreases the incidence and severity of brain injury associated with an episode of perinatal cerebral hypoxia-ischemia.     

Intracellular glucose concentration in derepressed yeast cells consuming glucose is high enough to reduce the glucose transport rate by 50%

1. This study describes the in vitro characterization of two potent and selective 5-HT6 receptor antagonists at the rat and human recombinant 5-HT6 receptor. 2. In binding assays with [3H]-LSD, 4-amino-N-(2,6 bis-methylamino-pyrimidin-4-yl)-benzene sulphonamide (Ro 04-6790) and 4-amino-N-(2,6 bis-methylamino-pyridin-4-yl)-benzene sulphonamide (Ro 63-0563) had mean pKi values +/-s.e.mean at the rat 5-HT6 receptor of 7.35+/-0.04 and 7.83+/-0.01, respectively and pKi values at the human 5-HT6 receptor of 7.26+/-0.06 and 7.91+/-0.02, respectively. 3 .Both compounds were found to be over 100 fold selective for the 5-HT6 receptor compared to 23 (Ro 04-6790) and 69 (Ro 63-0563) other receptor binding sites. 4. In functional studies, neither compound had any significant effect on basal levels of cyclicAMP accumulation in Hela cells stably expressing the human 5-HT6 receptor, suggesting that the compounds are neither agonists nor inverse agonists at the 5-HT6 receptor. However, both Ro 04-6790 and Ro 63-0563 behaved as competitive antagonists with mean +/-s.e.mean pA2 values of 6.75+/-0.07 and 7.10+/-0.09, respectively. 5. In rats habituated to observation cages, Ro 04-6790 produced a behavioural syndrome similar to that seen following treatment with antisense oligonucleotides designed to reduce the expression of 5-HT6 receptors. This behavioural syndrome consisted of stretching, yawning and chewing. 6. Ro 04-6790 and Ro 63-0563 represent valuable pharmacological tools for the identification of 5-HT6 receptors in natural tissues and the study of their physiological function.     

Interactions between N-methyl-D-aspartate receptor antagonists and the discriminative stimulus effects of morphine in rats

NMDA receptor antagonists have previously been reported to alter some pharmacological and behavioral effects of acute and chronic opioid administration. The present study assessed the interactions of NMDA antagonists with the discriminative stimulus properties of morphine. Adult male Long Evans rats were trained to discriminate 3.2 mg/kg of s.c. morphine from water under a two-lever fixed-ratio 10 schedule of food reinforcement. During test sessions. I.p. injections of the noncompetitive NMDA receptor antagonist dizocilpine (0.03-0.2 mg/kg), the competitive antagonists NPC 17742 (1-16 mg/kg), and SDZ 220-581 (0.1-3 mg/kg), the polyamine site antagonist eliprodil (3-17.3 mg/kg), the glycine-site partial agonist (+)-HA-966 (3-56 mg/kg), and the nonselective glutamate antagonist kynurenic acid (30-150 mg/kg) were coadministered with s.c. morphine (1-3.2 mg/kg; interaction tests) or water (generalization tests). In generalization tests, none of the compounds completely substituted for morphine. Concurrent administration of morphine and NMDA antagonists did not greatly alter the discriminative stimulus properties of morphine. Various doses of NPC 17742, SDZ 220-581, or (+)-HA-966 somewhat increased levels of morphine-appropriate lever selection, whereas some attenuation of morphine-lever selection was obtained when morphine was coadministered with eliprodil. These results show that NMDA antagonists have minimal interactions with the discriminative stimulus effects of morphine.     

Relapsing polychondritis

PURPOSE: This article describes a clinically-diagnosed case of relapsing polychondritis (RP), attended at the Hospital S?o Paulo, and presents a literature review of the subject. SOURCE OF RESEARCH: The literature review was made via Medline (1990-96). Lilacs (1980-96), textbooks of rheumatology, and some articles about the history of the disease. In Medline, 113 articles from 1990 to 1996 were found, and there were 23 articles from 1980 to 1996 in Lilacs. RESEARCH PROCEDURE: We reviewed the articles available at BIREME (Biblioteca Regional de Medicina) with the primary focus being on the disease in question. SUMMARY: RP is a rare disease of unknown etiology described initially by Jackson-Wartenhorst in 1923 and characterized by a recurrent and acute inflammatory process that causes the collapse of the cartilaginous structures and their subsequent replacement by fibrous connective tissue. The cartilage most commonly attacked is that of the auricle of the ear and nasal septum, while the cartilage of the trachea, larynx, epiglottis, ribs, and articulations may also be involved. Ocular inflammations and systemic reactions with fever are also described. In 1976, McAdam presented a complete prospective study of 23 patients, reviewed the 136 cases described up until that time, and then proposed diagnostic criteria which were later expanded by Damiani and Levine. Currently, more than 550 cases have been described. CONCLUSION: Although a rare disease, better knowledge of it is needed, as RP may be lethal with tracheal collapse and obstruction of respiratory pathways, making precise diagnosis and adequate therapeutic intervention necessary.     

Inhibition of tumor necrosis factor-alpha production by SK&F 98625, a CoA-independent transacylase inhibitor, in cultured rat peritoneal macrophages

When rat peritoneal macrophages were incubated in medium containing thapsigargin, tumor necrosis factor-alpha (TNF-alpha) production was increased time-dependently. In the presence of SK&F 98625, a CoA-independent transacylase inhibitor, the thapsigargin-induced TNF-alpha production was inhibited dose-dependently. Platelet-activating factor (PAF) and prostaglandin E2 (PGE2) production were also inhibited by SK&F 98625. The SK&F 98625-induced inhibition of TNF-alpha production was not prevented by addition of PGE2. PAF antagonists such as E6123, L-652,731 and CV-6209 partially inhibited the thapsigargin-induced TNF-alpha production, suggesting that concurrently produced PAF in thapsigargin-stimulated macrophages up-regulates TNF-alpha production. The inhibition by SK&F 98625 of thapsigargin-induced TNF-alpha production might be partly due to the inhibition of PAF production.     

Regulation of leukotriene and platelet-activating factor synthesis in human alveolar macrophages

It has been suggested that phospholipase A2 (PLA2) contributes to the regulation of leukotriene (LT) and platelet-activating factor (PAF) synthesis by controlling the release of their precursors, arachidonic acid (AA) and lysophosphatidylcholine (lysoPC), from membrane phospholipids. In rat alveolar macrophages (AMs), PLA2 appears to have a major role in LT synthesis but a more limited role in PAF synthesis. The present study was designed to define the role of PLA2 in LT and PAF synthesis in human AMs and determine whether differences exist between AMs obtained from normal subjects and those from patients with asthma. In the normal subjects, the calcium ionophore A23187 (Cal) increased AM PAF synthesis (percent incorporation of tritiated acetate) by 135% (p < 0.01) and LTB4 synthesis 88-fold (p < 0.001). Phorbol myristate acetate (PMA) had little effect alone, but it had a synergistic effect with Cal, increasing PAF synthesis by 466% and LTB4 synthesis to 229-fold above the control values (p < 0.001 for both). Ro 25-4331, a combined cytosolic (c) and secretory (s) PLA2 inhibitor, had little effect on the Cal-stimulated PAF synthesis, but it completely blocked the effect of PMA. It also blocked the Cal- and Cal+PMA-stimulated LTB4 synthesis. AACOCF3, a cPLA2 inhibitor, had no effect on either Cal or Cal+PMA-stimulated PAF synthesis. It reduced LTB4 synthesis, but it did so less effectively than Ro 25-4331. CoA-independent transacylase (CoAI-TA) activity in the AMs increased after stimulation and exposure to Ro 25-4331. SK&F 45905, a CoAI-TA inhibitor, reduced stimulated PAF synthesis by 30% to 40%. Patients with asthma had similar results except that cPLA2 had a greater role in stimulated LTB4 synthesis. These data indicate that PLA2 plays a direct role in human AM LT synthesis; both the cytosolic and secretory forms contribute to LT synthesis; PLA2 appears to have a more limited role in PAF synthesis, although it mediates the synergistic effect of PMA, probably via sPLA2; and CoAI-TA contributes to PAF synthesis during PLA2 inhibition. With the exception of the greater role for cPLA2 in stimulated LTB4 synthesis in the patients with asthma, the contributions of PLA2 and CoAI-TA to AM LT and PAF synthesis appear to be similar in normal subjects and patients with asthma.     

Cell-density-dependent expression of Cyp1a2 gene in monolayer-cultured adult mouse hepatocytes

The effects of PAF antagonists, of substances which influence the arachidonic acid metabolism, and of dexamethasone and ketotifen were evaluated in an acute PAF-induced mortality model in female NMRI mice. We established a dependence of sensitivity to PAF on strain (AB mice showed no dose dependence) and on sex of the animals as well as on the PAF charges used in our experiments. PAF produced resistance in surviving animals against the PAF-induced death on repeated application. The PAF antagonists, WEB 2170 and WEB 2086, provided the best dose-dependent protection against PAF toxicity, followed by dexamethasone, by the COX/LOX synthetase inhibitor X 86 (a BW 755 C-analogue) and by the PAF receptor antagonist BN 52021. Particularly remarkable was the excellent prevention by aspirin. Aspirin may not only inhibit the cyclooxygenase pathway but also endogenous PAF synthesis. Other drugs, i.e. indomethacin, the thromboxane receptor antagonist, BM 13177, the thromboxane synthetase inhibitor, HOE 944, as well as the lipoxygenase inhibitors (NDGA, esculetin, SHAM and phenidone) exerted a dose-dependent protection only at high doses.     

Role of platelet-activating factor in long-term potentiation of the rat medial vestibular nuclei

In rat brain stem slices, we investigated the role of platelet activating factor (PAF) in long-term potentiation (LTP) induced in the ventral part of the medial vestibular nuclei (MVN) by high-frequency stimulation (HFS) of the primary vestibular afferent. The synaptosomal PAF receptor antagonist, BN-52021 was administered before and after HFS. BN-52021 did not modify the vestibular potentials under basal conditions, but it reduced the magnitude of potentiation induced by HFS, which completely developed after the drug wash-out. The same effect was obtained by using CV-62091, a more potent PAF antagonist at microsomal binding sites, but with concentrations higher than those of BN-52021. By contrast both BN-52021 and CV-6209 had no effect on the potentiation once induced. This demonstrates that PAF is involved in the induction but not in the maintenance of vestibular long-term effect through activation of synaptosomal PAF receptors. In addition, we analyzed the effect of the PAF analogue, 1-O-hexadecyl-2-O- (methylcarbamyl)-sn-glycero-3-phosphocoline (MC-PAF) and the inactive PAF metabolite, 1-O-hexadecyl-sn-glycero-3-phosphocoline (Lyso-PAF) on vestibular responses. Our results show that MC-PAF, but not Lyso-PAF induced potentiation. This potentiation was prevented by D,L-2-amino 5-phosphonopentanoic acid, suggesting an involvement of N-methyl-D-aspartate receptors. Furthermore, under BN-52021 and CV-6209, the MC-PAF potentiation was reduced or abolished. The dose-effect curve of MC-PAF showed a shift to the right greater under BN-52021 than under CV-6209, confirming the main dependence of MC-PAF potentiation on the activation of synaptosomal PAF receptors. Our results suggest that PAF can be released in the MVN after the activation of postsynaptic mechanisms triggering LTP, and it may act as a retrograde messenger which activates the presynaptic mechanisms facilitating synaptic plasticity.     

Functional relevance of the expression of ligand-induced binding sites in the response to platelet GP IIb/IIIa antagonists in vivo

RGD-containing peptides and other antagonists of the platelet glycoprotein (GP) IIb/IIIa may induce a high-affinity binding site for fibrinogen and the expression of novel epitopes, called ligand-induced binding sites (LIBS). The functional relevance of LIBS expression in a canine model of coronary thrombolysis induced by tissue-type plasminogen activator (t-PA) was examined. Ro43-5054 (N-[N-[N-(p-amidinobenzoyl)-b-alanyl]-l-a-aspartyl]-3-phenyl-l- alanine) and Ro44-9883 ([1-(N-(p-amidinobenzoyl)-l-tyrosyl)-4-piperidinyl)oxy]acetic acid), antagonists of the GP IIb/IIIa receptor, were administered in increasing doses of 2 to 10 microg/kg/min, beginning 30 min before the infusion of t-PA. LIBS expression was determined by the binding of the monoclonal antibody, D3GP3, to platelets on exposure to Ro43-5054, Ro44-9883 and t-PA. Ro43-5054 was shown to induce LIBS, whereas Ro44-9883 and t-PA did not. Both drugs abolished platelet aggregation in response to U46619 and ADP ex vivo. Reocclusion was prevented with both Ro43-5054 and Ro44-9883, but neither drug altered reperfusion times (49 +/- 8 and 55 +/- 39 min). Both drugs increased the rate of bleeding compared with t-PA alone, but there was no difference in hemostasis between the two drugs. To determine whether the drugs differed in their effect on platelet activation in vivo, urinary 2,3-dinor-thromboxane (TX) B2, a major metabolite of TXB2, was determined by gas chromatography-mass spectrometry. After reperfusion, the urinary 2,3-dinor-TXB2 increased in the Ro43-5054-treated group, similar to control groups (32 +/- 8 and 37 +/- 9 ng/mg creatinine). This increase was blunted in the Ro44-9883-treated group (9 +/- 3 ng/mg creatinine). GP IIb/IIIa antagonists that do not induce LIBS result in a greater suppression of platelet activity but not in any discernible functional benefit in vivo.     

Can we rely on the family history?

Ginseng saponins and their degradation products have been screened for antagonist activity towards [3H]PAF (platelet activating factor) in washed rabbit platelet receptor binding studies. 20(S)- and delta20-ginsenosides Rg3, protopanaxadiol-type saponins, were found to be relatively potent PAF antagonists (IC50 = 4.9 x 10(-5) M and 9.2 x 10(-5) M, respectively).     

Induction of early gene expression in murine macrophages by synthetic lipid A analogs with differing endotoxic potentials

Numerous lipid A analogs have been synthesized in an attempt to dissociate endotoxic activities from beneficial immunomodulatory activities. In the present study, we have evaluated select lipid A analogs in macrophages for their ability to induce a panel of lipopolysaccharide (LPS)-inducible genes to gain insights into the molecular mechanisms which underlie endotoxicity. We evaluated three monosaccharide lipid A analogs: SDZ MRL 953, an agonist with an improved therapeutic margin over endotoxin; SDZ 281.288, a more toxic analog; and SDZ 880.431, an analog with proven LPS-inhibitory activity. In addition, three disaccharide lipid A analogs (i.e., lipid IVA, SDZ 880.611, and SDZ 880.924) that differ in acylation and phosphorylation patterns were also examined and compared with synthetic lipid A. With the exception of SDZ 880.431, each of these structurally diverse analogs was able to induce the complete panel of LPS-inducible genes, specifically genes which encode tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, 75-kDa type 2 TNF receptor (D7), IP-10, D3, and D8. These results underscore that macrophage stimulation by lipid A analogs is permissive to considerable structural diversity. Structures with favorable therapeutic indices (SDZ MRL 953, SDZ 880.611, and SDZ 880.924) were not different from structures with poor therapeutic indices (lipid A, lipid IVA, and SDZ 281.288) with regard to gene induction. Nonetheless, the nontoxic SDZ MRL 953 was approximately 1,000-fold less potent than synthetic lipid A at inducing TNF-alpha secretion, and perhaps this contributes to the lack of toxicity exhibited by this compound. The ability of compound SDZ 880.431 to inhibit TNF-alpha secretion induced by both SDZ MRL 953 and smooth LPS suggests that the monosaccharide and smooth LPS share a receptor or a portion thereof. A pattern of protein tyrosine phosphorylation similar to that induced by LPS was stimulated by the monosaccharide SDZ MRL 953 and SDZ 281.288 and disaccharides lipid IVA, SDZ 880.924, and SDZ 880.611, providing evidence for a common signalling pathway.     

Incidence and outcome of schizophrenia in whites, African-Caribbeans and Asians in London

The endogenous, meal-contingent release of bombesin (BN)-like peptides is thought to contribute to the termination of a meal. In the following experiments the potency of BN receptor antagonists to attenuate the ability of nutrients to suppress food intake was tested. First, the effectiveness of BN receptor subtype antagonists was verified by testing their ability to block the effects of exogenous BN on food intake. Rats were administered intraperitoneal (i.p.) injections of either saline or 0.1 mg/kg [D-Phe12,Leu14]BN (binds both GRP and NMB receptors), [D-Phe6]BN(6-13) ethyl amide (binds GRP > NMB), and cyclo-SS-octa (BIM-23042; binds NMB > GRP). Five minutes later rats were administered 8 micrograms/kg BN (i.p.) and milk intake was measured. Injections of [D-Phe12,Leu14]BN and [D-Phe6]BN(6-13) ethyl amide reliably attenuated the ability of BN to suppress milk intake whereas BIM-23042 was ineffective. The results show that the antagonists were behaviorally effective and that exogenous BN may exert its effects on food intake primarily through the GRP receptor subtype. Next, the antagonists were administered either 5 min prior to or 5 min after an intragastric nutrient load or no load in both overnight-deprived and nondeprived rats, and milk intake was then measured. Stomach loads reduced intake and this effect was not attenuated by BN receptor antagonists. Finally, rats were allowed to prefeed and the milk was then removed. Rats were then administered a BN receptor antagonist (0.1 and 1.0 mg/kg) or saline either immediately after the prefeed, 10 min later, or 20 min later. Milk diet was then returned and intake was measured. Peripheral injections of the BN receptor antagonist had no effect compared to saline on milk intake. Collectively, the results indicate that the blockade of peripheral Bn peptide receptors is not sufficient to attenuate the safety signals generated by stomach loads or prefeeding.     

Platelet-activating factor receptor stimulation disrupts neuronal migration In vitro

LIS-1 is a gene whose hemi-deletion causes the human neuronal migration disorder Miller-Dieker lissencephaly. It encodes a subunit of a brain platelet-activating factor (PAF) acetylhydrolase, an enzyme that inactivates PAF by hydrolyzing the acetyl moiety in the sn2 position of this phospholipid. Because PAF receptor activation has been shown to affect the developing neuronal cytoskeleton, we have hypothesized that a role for PAF in neurodevelopment is that of a modulator of neuroblast movement (a cytoskeletal function) and that an aberrant regulation of PAF could lead to an early arrest in migration. This report examines the effects of the nonhydrolyzable PAF receptor agonist methyl carbamyl PAF (mc-PAF) on the unidirectional in vitro migration of granule cells from cerebellar cell reaggregates on a laminin substrate. Bath treatment with mc-PAF yields a dose-dependent decrease in granule cell migration compared with controls. This effect can be blocked by the simultaneous bath application of BN 52021 and trans-BTD, PAF receptor-specific antagonists. Although mc-PAF minimally inhibited neurite growth, its primary effect was on somal movement along preextended neurites. These experiments suggest that the stimulation of neuronal PAF receptors could be one crucial step for the regulation of neuroblast migration and that disturbed PAF catabolism during neurodevelopment could contribute to the neuronal migration defects observed in Miller-Dieker lissencephaly.     

Candesartan (CV-11974) dissociates slowly from the angiotensin AT1 receptor

The mechanisms of the insurmountable antagonism of 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]1H-benzimid azole -7-carboxylic acid, candesartan (CV-11974), an angiotensin AT1 receptor antagonist, on angiotensin II-induced rabbit aortic contraction were examined in contraction and binding studies. Preincubation of the rabbit aorta with CV-11974 (0.1 nM) for 30 min reduced the maximal contractile response to angiostensin II by approximately 50%. This insurmountable antagonism of CV-11974 was reversed in the presence of losartan (1 microM), a surmountable angiotensin AT1 receptor antagonist. The inhibitory effect of CV-11974 on angiotensin II-induced contraction persisted longer after washing than did that of losartan but was irreversible. Scatchard analysis of [3H]CV-11974 binding in bovine adrenal cortical membranes indicated the existence of a single class of binding sites (Kd = 7.4 nM). Competition binding studies using angiotensin II receptor agonists and antagonists have demonstrated that [3H[CV-11974 binding sites may be identical to angiotensin AT1 receptors. The dissociation rate of [3H]CV-11974 binding (t1/2 = 66 min) was 5 times slower than that of [125I]angiotensin II binding (t1/2 = 12 min). These results suggest that the insurmountable antagonism by CV-11974 is due to its slow dissociation from angiotensin AT1 receptors.