Differential sensitivity of tumor targets to liver macrophage-mediated cytotoxicity.

Liver macrophages activated in vivo with bacterially derived lipopolysaccharide (LPS) display enhanced chemotaxis, phagocytosis, and oxidative metabolism. To determine if LPS also activates these mononuclear phagocytes for tumor cell killing, we compared the cytotoxic activity of macrophages from livers of rats treated with LPS (5 mg/kg, i.v.) with resident Kupffer cells. We found that both macrophage cell types displayed cytotoxicity towards rat N1S1 hepatoma and RBL-1 basophilic leukemia cells. Cytotoxicity of resident and LPS-activated liver macrophages towards these targets increased with cocultivation time, was dependent on the effector:target cell ratio, and appeared to involve extracellular lysis. No direct correlation between macrophage activation and cytotoxicity was observed towards these targets. While liver macrophages from LPS treated rats were more cytotoxic towards N1S1 cells, resident Kupffer cells were more cytotoxic towards RBL-1 cells. In further studies, resident Kupffer cells were also found to display extracellular cytolytic activity towards mouse P815 mastocytoma cells. In contrast, LPS-activated liver macrophage-mediated killing of these targets involved phagocytosis of intact tumor cells, as evidenced by light and electron microscopy and by uptake of 51Cr-labeled cells. These results suggest that cytotoxicity mediated by liver macrophages depends on the type of macrophage and the nature of the tumor cell target. In addition, cytotoxicity towards tumor targets appears to involve at least two different mechanisms including extracellular cytolysis and phagocytosis.

Effects of bile salts on the structural integrity of liposomes.

The effects of the bile salts, sodium taurocholate and sodium glycocholate on the structural integrity of liposomes of different lipid compositions were studied. The bile salt concentrations were in the range encountered in the human intestine and the incubations were conducted at 37 degrees C. Two measures of liposome integrity were used: (a) changes in their light-scattering properties; and (b) release of entrapped potassium chromate. The results show that "fluid" liposomes, i.e., those prepared from phospholipids whose phase transition temperatures are below 37 degrees C, were totally disrupted in the presence of 10 mM bile salts at pH 7.4, and their entire contents were released. In the case of liposomes prepared from phospholipids with phase transition temperatures above 37 degrees C, the effect of the bile salts became less pronounced as the phase transition temperature increased. Nevertheless, a considerable proportion of the entrapped marker compound was released from liposomes with phase transition temperatures as high as 60 degrees C. The relevance of these results to the reported liposome-mediated gastrointestinal uptake of insulin is discussed.

Platelet-activating factor-induced calcium mobilization and oxidative metabolism in hepatic macrophages and endothelial cells.

Fluorescence image analysis of the calcium sensitive dye Indo-1 was used to characterize platelet-activating factor (PAF)-induced calcium mobilization in hepatic macrophages and endothelial cells. PAF, but not lyso-PAF, an inactive analog, induced a rapid and transient increase in intracellular levels of calcium that appeared to depend on the presence of extracellular calcium. In both macrophages and endothelial cells, these effects were dose dependent, reaching maximal levels with 10 nM PAF. However, the kinetics of the calcium response to PAF in macrophages and endothelial cells was distinct. The endothelial cells responded more rapidly to PAF than the macrophages (within 1 min vs. 2-3 min, respectively) and displayed longer recovery periods (4 min vs. > 10 min, respectively). In contrast, the magnitude of the response to PAF was greater in the macrophages. In both cell types, triazolam and alprazolam, two PAF antagonists, and the calcium channel blocker verapamil inhibited PAF-induced calcium mobilization. PAF also stimulated superoxide anion production alone and in combination with the macrophage activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in both cell types. Macrophages produced more of this reactive oxygen intermediate in response to PAF and TPA than endothelial cells. Interestingly, in the absence of extracellular calcium or when verapamil was added to the cultures, superoxide anion production by the macrophages, but not the endothelial cells, was significantly reduced. These results demonstrate that, although hepatic macrophages and endothelial cells mobilize calcium in response to PAF, the characteristics of the response in each cell type are different. These differences may underlie, at least in part, distinct functional responses of the cells to PAF.

Induction of chemotaxis in mouse peritoneal macrophages by activators of protein kinase C.

Two activators of calcium and phospholipid dependent protein kinase (protein kinase C), the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), were compared as chemotactic agents for mouse peritoneal macrophages. Both of these compounds were found to induce chemotaxis in the macrophages to a similar extent in a time and dose dependent manner. Induction of chemotaxis was observed in the concentration range of 10-100 nM for TPA and 25-250 microM for OAG. Two structurally related synthetic sn 1,2-diacylglycerols, 1,2-dioctanoylglycerol (diC8) and 1,2-didecanoylglycerol (diC10), were also found to be chemotactic for macrophages, while monoacylglycerol (2-monoolein) was inactive. Of the diacylglycerols, OAG was found to be the most active followed by diC8 and diC10. In contrast to TPA, the synthetic diacylglycerols had no effect on superoxide anion release by the cells, suggesting that the mechanism of superoxide anion release by TPA in macrophages is distinct from chemotaxis. Phorbol-12,13-diacetate, a biologically inactive phorbol ester analog that inhibits the binding of TPA to its cellular receptors, inhibited macrophage chemotaxis induced by TPA, the synthetic diacylglycerols, and the complement fragment, C5a. Taken together, our results suggest that chemotaxis in macrophages may be mediated by activation of protein kinase C.

Distinct biochemical responses of hepatic macrophages and endothelial cells to platelet-activating factor during endotoxemia.

Acute endotoxemia is associated with activation of hepatic macrophages and endothelial cells. These cells release a variety of inflammatory mediators that have been implicated in tissue injury. In the present studies, we analyzed the biochemical responses of these cells to platelet-activating factor (PAF), a lipid autacoid released during hepatic inflammatory responses. To induce acute endotoxemia, rats were injected intravenously with lipopolysaccharide (LPS). Using the calcium sensitive fluorescent indicator dye Indo-1, we found that PAF induced a rapid and transient increase in intracellular calcium in both hepatic macrophages and endothelial cells. Induction of acute endotoxemia resulted in an increase in the amount of calcium mobilized by both cell types. Although endothelial cells from control rats were less responsive to PAF than macrophages, these cells were more sensitive to in vivo endotoxin. PAF was also found to cause a rapid decrease in intracellular pH in hepatic macrophages that was quantified by fluorescence image analysis using the pH sensitive dye SNAFL-calcein. This decrease occurred more rapidly in macrophages from endotoxemic rats. In cells from both control and endotoxemic rats, the effects of PAF on intracellular pH were inhibited by the specific PAF antagonist triazolam. In contrast to hepatic macrophages, PAF had no effect on intracellular pH in endothelial cells from either control or endotoxemic rats. Ligand binding studies demonstrated that both hepatic macrophages and endothelial cells possess high affinity binding sites for PAF. Macrophages expressed 6- to 7-fold more binding sites/cell than endothelial cells and exhibited a higher Kd. Whereas treatment of rats with LPS had no effect on the Kd for PAF binding to macrophages or on the number of binding sites, a significant increase in both of these receptor characteristics was observed in endothelial cells. Taken together, the present data suggest that the biochemical responses of endothelial cells and macrophages to PAF are distinct. Furthermore, cellular activation induced by PAF in endothelial cells appears to be independent of changes in intracellular pH.

Distinct patterns of nitric oxide production in hepatic macrophages and endothelial cells following acute exposure of rats to endotoxin.

Hepatic macrophages and endothelial cells play an important role in the clearance of endotoxin from the portal circulation. These cells are activated by endotoxin to release reactive mediators including superoxide anion, hydrogen peroxide, and nitric oxide, which have been implicated in hepatic inflammation and tissue injury. In the present studies we analyzed mechanisms regulating the production of nitric oxide by hepatic macrophages and endothelial cells following in vivo exposure to endotoxin. Rats were injected intravenously with Escherichia coli lipopolysaccharide (LPS, 5 mg/kg). Cells were isolated from the animals 48 h later by in situ perfusion of the liver with collagenase and pronase followed by differential centrifugation and centrifugal elutriation. We found that macrophages and endothelial cells from both untreated and endotoxin-treated rats readily synthesized nitric oxide following in vitro stimulation with interferon-gamma (IFN-gamma) and LPS alone and in combination. This response was dependent on l-arginine and was blocked by two nitric oxide synthase inhibitors, NG-monomethyl-l-arginine and l-canavanine. Macrophages produced more nitric oxide in response to LPS or LPS plus IFN-gamma than endothelial cells. In addition, nitric oxide production by both cell types in response to LPS plus IFN-gamma was increased after treatment of rats with endotoxin. Macrophages appeared to be more sensitive than endothelial cells to the in vivo effects of this inflammatory stimulus. Northern and Western blot analysis demonstrated that nitric oxide production by macrophages and endothelial cells in response to LPS plus IFN-gamma was due to increased expression of an inducible form of nitric oxide synthase (iNOS) mRNA and protein. Using fluorescence image analysis, iNOS protein was found to be localized in the cytoplasm of the cells. Treatment of rats with endotoxin was associated with increased expression of iNOS protein in the macrophages. The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also stimulated nitric oxide production by macrophages and endothelial cells from endotoxin-treated rats, although not as effectively as LPS and IFN-gamma. Macrophages were more responsive than endothelial cells to TPA. Furthermore, depletion of the cells of glutathione using buthionine sulfoximine had no major effect on nitric oxide production by macrophages but resulted in small but significant inhibition in endothelial cells. This suggests that this sulfhydryl-containing tripeptide does not regulate intracellular levels of reactive nitrogen intermediates in activated macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)

Lymphocyte-mediated nitric oxide production by rat endothelial cells.

Lymphocyte migration from the blood to sites of tissue injury is mediated, in part, through the interaction of these cells with endothelial cells lining the vessel walls. The ability of endothelial cells to produce nitric oxide may be important in this process. We found that the addition of the nonspecific lymphocyte activators lipopolysaccharide (LPS) or concanavalin A (Con A) to rat hepatic endothelial cell cultures from control or endotoxemic rats markedly enhanced the ability of these cells to produce nitric oxide. In contrast, wheat germ agglutinin (WGA) and phytohemagglutinin (PHA) had no effect on nitric oxide release. Coculture of endothelial cells with lymphocyte-rich preparations of rat thymocytes or splenocytes stimulated endothelial cell nitric oxide production. This response was enhanced by LPS or Con A and to a lesser extent by WGA or PHA. In contrast to endothelial cells, thymocytes and splenocytes did not produce nitric oxide either in the presence or absence of lymphocyte mitogens. Increased production of nitric oxide by endothelial cells in response to lymphocytes and lymphocyte mitogens was due, at least in part, to increased expression of protein for an inducible form of nitric oxide synthase, as measured by Western blotting. Stimulation of endothelial cell nitric oxide production by thymocytes and splenocytes was inhibitable by the specific nitric oxide synthase inhibitor NG-monomethyl-L-arginine and dependent on cell-cell contact. Thus, nitric oxide production by endothelial cells was reduced when the lymphocytes were physically separated from the endothelial cells using cell culture inserts. We hypothesize that nitric oxide released by endothelial cells increases vascular permeability, thereby allowing the extravasation of lymphocytes into the surrounding tissue, a process that may be important in inflammation, tissue injury, and/or wound healing.

Production of nitric oxide and peroxynitrite in the lung during acute endotoxemia.

Nitric oxide is a short-lived cytotoxic mediator that has been implicated in the pathogenesis of endotoxin-induced tissue injury and septic shock. In the present studies we determined whether this mediator is produced in the lung during acute endotoxemia. We found that intravenous injection of rats with bacterially derived lipopolysaccharide (LPS), a condition that induces acute endotoxemia, caused a time-dependent increase in inducible nitric oxide synthase (iNOS) mRNA expression in the lung, which reached a maximum after 24 h. This was correlated with nitric oxide production in the lung as measured by electron paramagnetic spin trapping, which was detectable within 6 h. Alveolar macrophages (AMs) and interstitial macrophages (IMs) isolated from rats 6-12 h after induction of acute endotoxemia were also found to exhibit increased nitric oxide production in response to in vitro stimulation with interferon-gamma (IFN-gamma) and LPS measured by nitrite accumulation in the culture medium. The effects of acute endotoxemia on nitric oxide production by these cells were, however, transient and returned to control levels by 24 h in AMs and 36 h in IMs. Interestingly, although nitrite accumulation in the culture medium of IMs isolated 48 h after induction of acute endotoxemia and stimulated with low concentrations of IFN-gamma and LPS was reduced, when compared with cells from control animals, these cells, as well as AMs, continued to express high levels of iNOS protein and mRNA. This was correlated with increased peroxynitrite production by the cells. Peroxynitrite has been shown to act as a nitrating agent and can generate nitrotyrosine residues in proteins. Using a specific antibody and immunohistochemistry, we found evidence of nitrotyrosine residues in sections of lungs 48 h after treatment of rats with endotoxin. These data suggest that nitric oxide produced by IMs and AMs can react with superoxide anion to form peroxynitrite. Taken together, the present studies demonstrate that AMs and IMs are activated following acute endotoxemia to produce reactive nitrogen intermediates and that both cell types contribute to inflammatory responses in the lung.

Mechanisms underlying reduced responsiveness of neonatal neutrophils to distinct chemoattractants.

Potential mechanisms underlying impaired chemotactic responsiveness of neonatal neutrophils were investigated. Two distinct chemoattractants were compared: bacterially derived N-formyl-methionyl-leucyl-phenylalanine (fMLP) and a unique chemotactic monoclonal antibody, designated DL1.2, which binds to a neutrophil antigen with an apparent molecular mass of 120 kDa. Chemotaxis of neutrophils toward fMLP, as well as DL1.2, was reduced in neonates when compared with adult cells. This did not appear to be a result of decreased fMLP receptor or DL1.2 antigen expression by neonatal neutrophils. fMLP, but not DL1.2, induced a rapid increase in intracellular calcium in adult and neonatal cells, which reached a maximum within 30 s. The calcium response of cells from neonates to fMLP was reduced when compared with adult cells, and an unresponsive subpopulation of neonatal neutrophils was identified. NF-kappaB nuclear binding activity induced by fMLP and DL1.2, as well as expression of the p65 NF-kappaB subunit and IkappaB-alpha, was also significantly reduced in neonatal cells, when compared with adult cells. In contrast, although fMLP, but not DL1.2, activated p42/44 and p38 mitogen-activated protein (MAP) kinases in neutrophils, no differences were observed between adults and neonates. Chemotaxis of adult and neonatal neutrophils toward fMLP and DL1.2 was also blocked to a similar extent by inhibitors of phosphatidylinositol 3-kinase, as well as an inhibitor of NF-kappaB. These findings indicate that reduced chemotactic responsiveness in neonatal neutrophils is a result of, at least in part, aberrations in chemoattractant-induced signaling. However, the biochemical pathways mediating this defect appear to be related to the specific chemoattractant.

Prooxidant and antioxidant functions of nitric oxide in liver toxicity.

In response to tissue damage and inflammation induced by a variety of xenobiotics including acetaminophen, carbon tetrachloride, ethanol, galactosamine, and endotoxin, as well as disease states such as viral hepatitis, and postischemic and regenerative injury, the liver produces large quantities of nitric oxide. Indeed, nearly all cell types in the liver including hepatocytes, Kupffer cells, stellate cells, and endothelial cells have the capacity to generate nitric oxide. Thus, these cells, as well as infiltrating leukocytes, may indirectly augment tissue injury. In many models of liver damage, nitric oxide and its oxidation products such as peroxynitrite contribute to the injury process by directly damaging the tissue or by initiating additional immunologic reactions that result in damage. In some models, nitric oxide donors or peroxynitrite can mimic the cytotoxic actions of liver toxins. Moreover, agents that prevent the generation of nitric oxide or antioxidants that bind reactive nitrogen intermediates, or knockout mice with reduced capacity to produce nitric oxide, are protected from xenobiotic-induced tissue injury. In contrast, there have been reports that blocking nitric oxide production enhances xenobiotic-induced tissue injury. This has led to the concept that nitric oxide either inactivates proteins critical for xenobiotic-induced tissue injury or acts as an antioxidant, reducing cellular levels of cytotoxic reactive oxygen intermediates. Whether or not nitric oxide or secondary oxidants generated from nitric oxide act as mediators of tissue injury or protect against toxicity is likely to depend on the precise targets of these reactive nitrogen intermediates, as well as levels of superoxide anion present and the extent to which tissue injury is mediated by reactive oxygen intermediates. In addition, as toxicity is a complex process involving a variety of cell types and many soluble mediators, the contribution of each of these factors must be taken into account when considering the role of nitric oxide as a determinant of tissue injury.

The use of TNF family ligands and receptors and agents which modify their interaction as therapeutic agents.

The earlier known TNF family cytokines have fairly wide physiological actions, mainly in inflammation and immune responses. It is now considered feasible to develop these large proteins themselves as therapeutic agents, but in addition, modular organisation of structures of biological proteins, and the identification of localised ligand-receptor interaction sites, allow rational design of smaller, preferably nonpeptide molecules which interfere with these protein:protein interactions. Neutralising anti-TNF antibodies and soluble TNF receptor preparations were shown to have striking anti-inflammatory activities in clinical studies, particularly in rheumatoid arthritis. As the TNF beta:TNFR1 co-crystal structure was the first in the family to be solved, rational drug design based on the ligand:receptor interaction sites is more advanced. Ligand mutations and a peptide sequence from TNF-alpha have given useful information regarding ligand-receptor interactions. Small peptide sequences from TNFR I which interact with the ligand have shown some activity in blocking the biological actions of TNF. The physiological activities of several recently-discovered ligands are more limited, giving possibilities for selective treatment of several diseases. For example, TRAIL can induce apoptosis in a wide range of tumour cells with little effects on normal tissues, both in vitro and in vivo. The co-crystal structure of TRAIL with one of its signalling receptors TRAILR 2 has been solved, opening the way to rational small molecule drug design. TRANCE (RANKligand) has modulatory effects on the dendritic cell:T cell interaction in immune responses. However, it plays a more major controlling role in the development of osteoclasts and their bone resorbing activity. In this way, it is a very interesting drug development target for the treatment of bone disorders such as osteoporosis. A recombinant secreted inhibitory receptor, osteoprotegerin (OPG), is in Phase 1 clinical trial for the treatment of hyper-resorptive bone diseases. However, OPG also blocks TRAIL and may not be sufficiently specific in long term therapy, but it is hoped that inhibitors of the interaction of TRANCE and its specific signalling receptor, RANK, can be rationally designed.

Receptor sub-types involved in responses of Purkinje cell to exogenous excitatory amino acids and local electrical stimulation in cerebellar slices in the rat.

The effects of the NMDA receptor antagonist, 2-amino-5-phosphonovalerate (APV) and non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on responses of Purkinje cells to exogenous excitatory amino acids and to electrical stimulation of the parallel fibres, were investigated in slices of the cerebellum of the rat. Glutamate, aspartate, kainate and quisqualate all induced excitation of Purkinje cells. Responses to kainate and quisqualate were blocked by CNQX (10 microM) but not by APV (10 microM). N-Methyl-D-aspartate induced biphasic excitatory-inhibitory responses, both components of which were blocked by APV but not by CNQX. The inhibitory component was less sensitive to blockade by APV but was totally blocked by bicuculline, the GABAA receptor antagonist. Parallel fibre stimulation most commonly induced inhibition of Purkinje cells, with or without preceding excitation. This inhibition was blocked by APV and excitatory responses were often revealed. A less commonly-observed predominantly excitatory response was blocked by CNQX but not by APV and inhibition tended to be revealed. These data suggest that parallel fibre-Purkinje cell synapses possess non-NMDA postsynaptic receptors, while the parallel fibre-inhibitory interneuron synapses possess functional NMDA receptors.

Reduction of cerebellar GABAA responses by interleukin-1 (IL-1) through an indomethacin insensitive mechanism.

Recently, a role of IL-1 in the central nervous system has been described, principally a fever-inducing effect in the hypothalamus through a prostaglandin second messenger system. IL-1 has also been shown to potentiate gamma-aminobutyric acid (GABAA) responses in embryonic chick neurones. This study describes the investigation of the effect of IL-1 on GABAA responses within the in vitro rat cerebellar slice, a preparation containing intact neuronal circuitry. Stimulation of the area of passage of paralleled fibres produced a pure GABAA inhibition of the spontaneous firing of Purkinje cells. 5 and 10 ng/ml IL-1 produced a reduction in the duration of inhibition 10 min after beginning perfusion of IL-1. This effect reversed within 15 min of washing out the IL-1. 10 ng/ml IL-1 also reduced the effects of exogenously-applied GABA (0.1 mM) with the same time course. In the presence of 1 uM indomethacin, there was no change in the effect of the IL-1. It can therefore be concluded that the reduction in cerebellar GABAA responses by IL-1 is not mediated by the indomethacin-sensitive prostaglandin second messenger system.

The behavioural effects of RU 24969, a suggested 5-HT1 receptor agonist in rodents and the effect on the behaviour of treatment with antidepressants.

Some behavioural and biochemical consequences of the administration of RU 24969 (5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole), a drug which has been shown to be a potent displacing agent at the 5-HT1 binding site, have been studied in mice and rats. In both species RU 24969 produced a dose-dependent increase in locomotor activity. No head-weaving, head-twitching or reciprocal forepaw treading (behaviour suggested to be 5-HT2 receptor mediated) were seen, nor did the body temperature increase p-Chlorophenylalanine did not alter either the behaviour or its intensity. Pretreatment with both methysergide (10 mg/kg) and metergoline (2.5 mg/kg) enhanced the behavioural response in rats, whilst haloperidol (100 micrograms/kg) inhibited the response. Propranolol caused a small and variable decrease in the increase in locomotion induced by RU 24969 in both mice and rats. The drug RU 24969 (10 mg/kg) inhibited the rate of synthesis of 5-HT in rat brain by about 50%. Pretreatment of rats with desmethylimipramine over a longer term or clenbuterol given acutely, treatments known to enhance the behavioural responses of rats to various other 5-HT agonists, did not alter the RU 24969-induced response. Repeated administration of electroconvulsive shock, however, did produce an enhanced locomotor response to RU 24969. The results obtained are consistent with the view that administration of 5-HT agonists, such as quipazine and 5-methoxy-N,N-dimethyltryptamine, produces behaviour that results from stimulation of both 5-HT1 and 5-HT2 receptor populations.

The activity of cis and trans -2- (aminomethyl) cyclopropane carboxylic acids on GABA - related mechanisms in the central nervous system of Limulus and Periplanata and of mammals.

Some pharmacological actions of (+/-) - cis - and (+/-) - trans -2-(aminomethyl) cyclopropane carboxylic acids, conformationally restricted analogues of GABA, are described. The trans isomer (TAMP) was 17 and 4.5 times less active than GABA in hyperpolarising central neurones of Limulus and Periplanata and 10.8 times less active in displacing [3H]-muscimol from rat brain membranes. The cis isomer (CAMP) was ineffective on Limulus neurones at 35 mumoles, 650 times less active on Periplanata neurones and only weakly displaced [3H]-muscimol at 100 microM. Neither isomer inhibited synaptosomal uptake of [3H]-GABA at 100 microM. These data suggest that the invertebrate receptors studied respond to GABA in an extended conformation.

2-(oxadiazolyl)- and 2-(thiazolyl)imidazo[1,2-a]pyrimidines as agonists and inverse agonists at benzodiazepine receptors.

Oxadiazoles, like the benzoyl group in a series of imidazo[1,2-a]pyrimidines, have been found to be metabolically stable alternatives to ester groups in benzodiazepine-receptor ligands. This change has lead to a number of compounds which bind to the receptors and which exhibit potent agonist activity in a food-motivated conflict test thought to predict anxiolytic properties. Compounds 4, 5, and 13 were equipotent with chlordiazepoxide but showed little or no myorelaxant effects. Replacing the oxadiazole group by thiazole gave compounds such as 23 which binds to the benzodiazepine receptor but exhibits the intrinsic activity of a partial inverse agonist in vivo.

(Imidazo[1,2-a]pyrimidin-2-yl)phenylmethanones and related compounds as potential nonsedative anxiolytics.

Several series of heterocyclic carboxylic esters were found to be active in the benzodiazepine receptor binding assay, a typical example being ethyl 7-ethyl-5-methoxyimidazo[1,2-a]quinoline-2-carboxylate (4b) with an IC50 of 150 nM. The corresponding phenylmethanone 5d was more potent with an IC50 of 14 nM and was orally active in animal models thought to predict anxiolytic effects. The synthesis of a large number of compounds resulted in the optimization of this activity in a series of (imidazo[1,2-a]pyrimidin-2-yl)phenylmethanones of which compounds 7e, 8b, 8h, 8j, and 8k were equipotent with chlordiazepoxide while exhibiting reduced anticonvulsant activity, little or no muscle relaxation, and negligible sedative effects.

Central effects of nicotinamide and inosine which are not mediated through benzodiazepine receptors.

The actions of nicotinamide and inosine were investigated on rat cerebellar Purkinje cells using ionophoretic and extracellular recording techniques. Ionophoretic application of nicotinamide or inosine showed that they were potent inhibitors of Purkinje cell firing. This inhibition differed from that induced by benzodiazepines in that it was not reversed by the GABA antagonists bicuculline methiodide and picrotoxin. RO 15-1788, the specific benzodiazepine antagonist, did not reverse the effects of nicotinamide. Chlordiazepoxide has been shown to increase significantly social interaction between pairs of male rats and this increase can be reversed by RO 15-1788, 20 mg kg-1 i.p. Nicotinamide also caused a small increase in social interaction but this effect was not reversed by the benzodiazepine antagonist. Inosine did not increase social interaction. [3H]-flunitrazepam binding studies showed that nicotinamide and inosine have only low affinities for the benzodiazepine binding site. These results suggest that while nicotinamide may exert some neuronal depressant and anxiolytic activity, its site of action appears not to be associated with the benzodiazepine receptor site. Similarly, inosine exerts a neuronal depressant effect dissimilar from that of benzodiazepines.

Minimal amidine structure for inhibition of nitric oxide biosynthesis.

Pharmacological modulation of nitric oxide synthase activity has been achieved using structural analogs of arginine. In the present studies, we demonstrated that the minimal amidine structure required for enzymatic inhibition is formamidine. We found that the production of nitric oxide by primary cultures of rat hepatocytes and several mouse and human cell lines, including RAW 264.7 macrophages, PAM 212 keratinocytes, G8 myoblasts, S180 sarcoma, CX-1 human colon cells, and GH3 rat pituitary cells, was inhibited in a concentration- and time-dependent manner by formamidine. Formamidine was 2- to 6-fold more effective in inhibiting nitric oxide production in cells expressing inducible nitric oxide synthase (NOS2) than in a cell line expressing calcium-dependent neuronal nitric oxide synthase (NOS1). Whereas formamidine had no effect on gamma-interferon-induced expression of nitric oxide synthase protein, its enzymatic activity was blocked. Kinetic analysis revealed that formamidine acts as a simple competitive inhibitor with respect to arginine (K(i) formamidine approximately 800 microM). Using a polarographic microsensor to measure real-time flux of nitric oxide release from RAW 264.7 macrophages, formamidine was found to require 30-90 min to inhibit enzyme activity, suggesting that cellular uptake of the drug may limit its biological activity. Our data indicate that formamidine is an effective inhibitor of nitric oxide production. Furthermore, its low toxicity may make it useful as a potential therapeutic agent in diseases associated with the increased production of nitric oxide.

Potential and limitations of drug targeting: an overview.

Potential and limitations of current efforts in drug targeting are discussed in terms of the criterion that targeting strategies must ultimately be gauged by their success in producing selectivity of pharmacological response thereby reducing or eliminating side effects which are not mechanism-related. A wide variety of approaches are considered, including: local drug administration, differential metabolism, carriers and vehicles, macromolecular recognition, site-specific activation and molecular specificity. Each approach is briefly assessed for its potential to be developed as a realistic candidate for human health care.