Novel phenylpyrimidine derivatives containing a hydrazone moiety protect rice seedlings from injury by metolachlor.

One strategy for solving the phytotoxicity of herbicides is to apply herbicide safeners that can efficiently alleviate the injuries of agricultural crops caused by herbicides. When metolachlor, a chloroacetamide herbicide, is applied with paddy rice, for example, the mechanisms associated with metolachlor and its residue negatively impact on the growth and yields of rice. To identify novel high-activity herbicide safener candidates for metolachlor, a series of (E)-4-(2-substituted hydrazinyl)-6-chloro-2-phenyl pyrimidines were synthesized and their structures were confirmed using IR (infrared radiation), 1H NMR, 13C NMR, and HRMS (high resolution mass spectrometry). The herbicide safener activities were then evaluated via primary tests. Compounds 3i and 3t were found to have the best herbicide activity on plant height. These compounds were then further screened for their activities at lower concentrations and showed better or similar activities compared to the positive control fenclorim, a commercial herbicide safener. The compounds 3i and 3t significantly enhanced glutathione S-transferase (GST) activity related with the herbicide safener activity in both shoots and roots tissues. Moreover, a qPCR (Real-time quantitative polymerase chain reaction) analysis found that the 3i and 3t treatments enhanced the expressions of OsGSTU3, OsGsTU39, and OsGSTF5. Finally, the results of an acute toxicity assessment with zebrafish (Danio rerio) embryos using treatments 3i and 3t indicated they are relatively safe to aquatic organisms.

A Chemical Proteomic Probe for the Mitochondrial Pyruvate Carrier Complex.

Target engagement assays are crucial for establishing the mechanism-of-action of small molecules in living systems. Integral membrane transporters can present a challenging protein class for assessing cellular engagement by small molecules. The chemical proteomic discovery of alpha-chloroacetamide (αCA) compounds that covalently modify cysteine-54 (C54) of the MPC2 subunit of the mitochondrial pyruvate carrier (MPC) is presented. This finding is used to create an alkyne-modified αCA, YY4-yne, that serves as a cellular engagement probe for MPC2 in click chemistry-enabled western blotting or global mass spectrometry-based proteomic experiments. Studies with YY4-yne revealed that UK-5099, an alpha-cyanocinnamate inhibitor of the MPC complex, engages MPC2 with remarkable selectivity in human cells. These findings support a model where UK-5099 inhibits the MPC complex by binding to C54 of MPC2 in a covalent reversible manner that can be quantified in cells using the YY4-yne probe.

The cognition-enhancing activity of E1R, a novel positive allosteric modulator of sigma-1 receptors.

BACKGROUND AND PURPOSE: Here, we describe the in vitro and in vivo effects of (4R,5S)-2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-acetamide (E1R), a novel positive allosteric modulator of sigma-1 receptors. EXPERIMENTAL APPROACH: E1R was tested for sigma receptor binding activity in a [³H](+)-pentazocine assay, in bradykinin (BK)-induced intracellular Ca²âº concentration ([Ca²âº](i)) assays and in an electrically stimulated rat vas deferens model. E1R's effects on cognitive function were tested using passive avoidance (PA) and Y-maze tests in mice. A selective sigma-1 receptor antagonist (NE-100), was used to study the involvement of the sigma-1 receptor in the effects of E1R. The open-field test was used to detect the effects of E1R on locomotion. KEY RESULTS: Pretreatment with E1R enhanced the selective sigma-1 receptor agonist PRE-084's stimulating effect during a model study employing electrically stimulated rat vasa deferentia and an assay measuring the BK-induced [Ca²âº](i) increase. Pretreatment with E1R facilitated PA retention in a dose-related manner. Furthermore, E1R alleviated the scopolamine-induced cognitive impairment during the PA and Y-maze tests in mice. The in vivo and in vitro effects of E1R were blocked by treatment with the selective sigma-1 receptor antagonist NE-100. E1R did not affect locomotor activity. CONCLUSION AND IMPLICATIONS: E1R is a novel 4,5-disubstituted derivative of piracetam that enhances cognition and demonstrates efficacy against scopolamine-induced cholinergic dysfunction in mice. These effects are attributed to its positive modulatory action on the sigma-1 receptor and this activity may be relevant when developing new drugs for treating cognitive symptoms related to neurodegenerative diseases.

Anxiolytic effects of the melatonin MT(2) receptor partial agonist UCM765: comparison with melatonin and diazepam.

Melatonin (MLT) is a neurohormone known to be involved in the regulation of anxiety. Most of the physiological actions of MLT in the brain are mediated by two high-affinity G-protein-coupled receptors, denoted MT(1) and MT(2). However, the particular role of these receptors in anxiety remains to be defined. Here we used a novel MT(2)-selective partial agonist, UCM765 to evaluate the involvement of MT(2) receptors in anxiety. Adult male rats were acutely injected with UCM765 (5-10-20mg/kg), MLT (20mg/kg) or diazepam (DZ, 1mg/kg). Anxiety-related behaviors were assessed in the elevated plus maze test (EPMT), novelty suppressed feeding test (NSFT) and open field test (OFT). UCM765 at the dose of 10mg/kg showed anxiolytic-like properties by increasing the time spent in the open arm of the EPMT, and by reducing the latency to eat in a novel environment in the NSFT. In the EPMT, animals treated with UCM765 (10mg/kg) or MLT (20mg/kg) spent more time in the open arms compared to vehicle-treated animals, but to a lesser extent compared to DZ (1mg/kg). In the NSFT, all treatments similarly decreased the latency to eat in a novel environment compared to vehicle. UCM765 and MLT did not affect the total time and the number of entries into the central area of the OFT, but unlike DZ, did not impair locomotion. The anxiolytic effects of UCM765 and MLT in the EPMT and the NSFT were blocked using a pre-treatment with the MT(1)/MT(2) antagonist luzindole (10mg/kg) or the MT(2) antagonist 4P-PDOT (10mg/kg). These results demonstrated, for the first time, the anxiolytic properties of UCM765 and suggest that MT(2)-receptors may be considered a novel target for the development of anxiolytic drugs.

O antagonismo com acetamida em experimentos com ovinos, caprinos e coelhos indica monofluoroacetato como princípio tóxico de Pseudocalymma elegans Bignoniaceae / Antagonism of acetamid in experiments with sheep, goats and rabbits indicates that monofluoroacetate is the toxic principle of Pseudoca lymma elegans Bignoniaceae

O presente trabalho teve como objetivo avaliar o efeito protetor da acetamida nas intoxicações experimentais por Pseudocalymma elegans em ovinos, caprinos e coelhos, com a finalidade de comprovar indiretamente que o monofluoroacetato (MF) é responsável pelos sinais clínicos e a morte dos animais que ingerem essa planta. Foram realizados experimentos para determinar a dose letal da planta coletada em Rio Bonito, RJ, em diferentes épocas do ano para ovinos e caprinos e ajustar a dose de acetamida a ser administrada. - No primeiro experimento, dois ovinos e dois caprinos receberam 1,0g/kg de P. elegans fresca e um animal de cada espécie foi tratado previamente com 2,0g/kg de acetamida. Nenhum animal apresentou alterações clínicas ou morreu. Ao que tudo indica a planta poderia estar menos tóxica, já que foi coletada no fim da estação das águas. - No segundo experimento, dois ovinos e dois caprinos receberam 0,67 e 1,0g/kg da planta dessecada, após tratamento prévio, com 2,0 e 3,0g/kg de acetamida, respectivamente. Todos os animais morreram, pois administramos doses muito altas de P. elegans. - No terceiro experimento, dois ovinos e dois caprinos receberam 0,333g/kg de P. elegans dessecada, após administração prévia de 2,0 g/kg de acetamida. Uma semana depois, o protocolo acima foi repetido, porém sem o antídoto. Nos experimentos com coelhos, foram administradas doses de 0,5 e 1,0g/kg de elegans dessecada após a administração de 3,0g/kg de acetamida. Sete dias depois, repetiu-se o protocolo, com exceção da administração de acetamida. Esta, quando administrada previamente, evitou o aparecimento dos sinais clínicos e a morte dos ovinos, caprinos e coelhos, já os animais não tratados com acetamida apresentaram sintomatologia e morreram. Clinicamente, os ovinos e caprinos manifestaram taquicardia, jugulares ingurgitadas, pulso venoso positivo, decúbito esternal e tremores musculares. Na "fase dramática", os animais caíam em decúbito lateral, esticavam ...

This study aimed to evaluate the protective effect of acetamid in experimental poisoning by Pseudocalymma elegans in sheep, goats and rabbits, in order to prove indirectly that monofluoroacetate (MF) is responsible for the clinical signs and death of animals that ingested the plant. Experiments were performed to determine for sheep and goats the lethal dose of P. elegans collected in Rio Bonito, RJ, in different seasons, and to adjust the dose of acetamid to be administered. - In the first experiment, two sheep and two goats received 1.0g/kg of fresh P. elegans, and two (one sheep and one goat) were pretreated with 2.0g/kg of acetamid. None of the animals showed clinical signs or died. Possibly, the plant could be less toxic, since it was collected at the end of the rainy season. - In the second experiment, two sheep and two goats received 0.67 and 1.0g/kg of the dried plant, after pretreatment with 2.0 and 3.0g/kg of acetamid, respectively. All animals died, as the administered doses of P. elegans were very high. - In the third experiment, two sheep and two goats received 0.333g/kg of dried P. elegans after previous administration of 2.0g/kg of acetamid; a week later, the protocol above was repeated, but without the antidote. In experiments with rabbits, doses of 0.5 and 1.0g/kg of dried P. elegans were given after administration of 3.0g/kg of acetamid; seven days later, the same protocol was repeated, except the administration of acetamide. This procedure, when acetamid was administered before, prevented the appearance of clinical signs and death of sheep, goats and rabbits. But the animals not treated with acetamid showed symptoms of poisoning and died. Clinically, the sheep and goats had tachycardia, engorged jugular vein, positive venous pulse, lateral recumbence, and muscle tremors. In the "dramatic phase", the animals fell into lateral position, stretched the limbs, were paddling and died within minutes. The rabbits showed apathy, ...

Cardioprotective effect of a dual acting epoxyeicosatrienoic acid analogue towards ischaemia reperfusion injury.

BACKGROUND AND PURPOSE: Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid that are metabolized into dihydroxyepoxyeicosatrienoic acids (DHET) by soluble epoxide hydrolase (sEH). The current investigations were performed to examine the cardioprotective effects of UA-8 (13-(3-propylureido)tridec-8-enoic acid), a synthetic compound that possesses both EET-mimetic and sEH inhibitory properties, against ischaemia-reperfusion injury. EXPERIMENTAL APPROACH: Hearts from C57BL/6 mice were perfused in Langendorff mode and subjected to ischaemia reperfusion. Mechanistic studies involved co-perfusing hearts with either 14,15-EEZE (a putative EET receptor antagonist), wortmannin or PI-103 (class-I PI3K inhibitor). H9c2 cells were utilized to investigate the protective effects against mitochondrial injury following anoxia reoxygenation. KEY RESULTS: Perfusion of UA-8 significantly improved postischaemic left ventricular developed pressure (LVDP) and reduced infarction following ischaemia reperfusion compared with control and 11,12-EET. UA-7 (13-(2-(butylamino)-2-oxoacetamido)tridec-8(Z)-enoic acid), a compound lacking sEH inhibitory properties, also improved postischaemic LVDP, while co-perfusion with 14,15-EEZE, wortmannin or PI-103 attenuated the improved recovery. UA-8 prevented anoxia-reoxygenation induced loss of mitochondrial membrane potential and cell death in H9c2 cells, which was blocked by co-treatment of PI-103. CONCLUSIONS AND IMPLICATIONS: UA-8 provides significant cardioprotection against ischaemia reperfusion injury. The effects are attributed to EETs mimetic properties, which limits mitochondrial dysfunction via class-I PI3K signalling.

Agomelatine, a melatonin receptor agonist with 5-HT(2C) receptor antagonist properties, protects the developing murine white matter against excitotoxicity.

Periventricular leukomalacia is a major cause of cerebral palsy. Perinatal white matter lesions associated with cerebral palsy appears to involve glutamate excitotoxicity. When injected intracerebrally into newborn mice, the glutamatergic analog, ibotenate, induces white matter cysts mimicking human periventricular leukomalacia. Intraperitoneal injection of melatonin was previously shown to be neuroprotective in this mouse model. The goal of the present study was to compare in this model the protective effects of agomelatine (S 20098), a melatonin derivative, with melatonin. Mice that received intraperitoneal S 20098 or melatonin had significant reductions in size of ibotenate-induced white matter cysts when compared with controls. Although agomelatine and melatonin did not prevent the initial appearance of white matter lesions, they did promote secondary lesion repair. Interestingly, while melatonin effects were only observed when given within the first two hours following the excitotoxic insult, agomelatine was still significantly neuroprotective when administered eight hours after the insult. The protective effects of agomelatine and melatonin were counter-acted by co-administration of luzindole or S 20928, two melatonin receptor antagonists. Agomelatine, acting through melatonin receptors, could represent a promising new drug for treating human periventricular leukomalacia and have beneficial effects on neuroplasticity.

Organ-specific expression of glutathione S-transferases and the efficacy of herbicide safeners in Arabidopsis.

The functions of plant glutathione S-transferases (GSTs) under normal growth conditions are poorly understood, but their activity as detoxification enzymes has been harnessed in agriculture for selective weed control. Herbicide safeners protect monocot crops from herbicide injury but have little effect on weedy monocot or dicot species. Protection by safeners is associated with expression of herbicide-metabolizing enzymes including GSTs, but the basis for selective action of safeners between monocots and dicots is not known. To address this question we have studied the response of Arabidopsis (Arabidopsis thaliana) to various safeners. Benoxacor, fenclorim, and fluxofenim did not protect Arabidopsis from herbicide injury but did induce RNA expression of the glutathione-conjugate transporters encoded by AtMRP1, AtMRP2, AtMRP3, and AtMRP4. These safeners also induced the organ-specific expression of AtGSTU19 and AtGSTF2, two previously characterized Arabidopsis GSTs from different classes of this enzyme family. RNA hybridization, immunoblot, and reporter gene analyses indicated expression of AtGSTU19 induced by safeners predominated in roots. To test the hypothesis that increased expression of AtGSTU19 would be sufficient to provide tolerance to chloroacetamide herbicides, a chimeric gene was produced containing the open reading frame for this GST driven by a constitutive promoter. Plants containing this transgene had a modest increase in AtGSTU19 protein, predominantly in roots, but this had no effect on tolerance to chloroacetamide herbicides. The localized induction of GSTs by safeners in roots of Arabidopsis may explain why these compounds are unable to provide herbicide tolerance to dicot plant species.

Anxiolytic-like activity of agomelatine and melatonin in three animal models of anxiety.

The activity of the novel antidepressant agomelatine was evaluated in three models of anxiety and compared with that of melatonin and two anxiolytics, diazepam and buspirone. All drugs were tested 2 h before and 2 h after the dark phase of the diurnal cycle. Morning and evening agomelatine (10-75 mg/kg) administration increased animals' responses in the elevated plus maze and Vogel tests. Melatonin (10-75 mg/kg) enhanced open arms exploration in the evening experiment and was inactive in the Vogel test. In the conditioned ultrasonic vocalization test, agomelatine, but not melatonin, was active in the morning and evening experiment. Melatonin antagonist, S22153 (20 mg/kg), enhanced the action of morning and evening agomelatine administration in the Vogel and conditioned ultrasonic vocalization tests, while in the elevated plus maze test, S22153 inhibited effects of evening but not morning melatonin and agomelatine administration. These results indicate the involvement of both the melatonin and the 5-HT2C receptors in the mechanism of anxiolytic-like action of agomelatine.

Effects of chronic hypoxia on MK-801-induced changes in the acute hypoxic ventilatory response.

Chronic hypoxia increases the sensitivity of the central nervous system to afferent input from carotid body chemoreceptors. We hypothesized that this process involves N-methyl-D-aspartate (NMDA) receptor-mediated mechanisms and predicted that chronic hypoxia would change the effect of the NMDA receptor blocker dizocilpine (MK-801) on the poikilocapnic hypoxic ventilatory response (HVR). Male Sprague-Dawley rats were studied before and after acclimatization to hypoxia (70 Torr inspiratory Po(2) for 9 days). We measured ventilation (VI) and the HVR before and after systemic MK-801 treatment (3 mg/kg ip). MK-801 resulted in a constant respiratory frequency (approximately 175 min(-1)) during acute exposure to 10% and 30% O(2) before and after acclimatization. MK-801 had no effect on tidal volume (VT) before acclimatization, but it significantly decreased Vt when the animals were breathing 10% O(2) after acclimatization. The net effect of MK-801 was to eliminate the O(2) sensitivity of Vi before (via changes in respiratory frequency) and after (via changes in VT) acclimatization. Hence, chronic hypoxia altered the effect of MK-801 on the acute HVR, primarily because of increased effects on Vt. This indicates that changes in NMDA receptor-mediated neurotransmission may be involved in ventilatory acclimatization to hypoxia. However, further experiments are necessary to determine the precise location of such plasticity in the central nervous system.

Chemopreventive properties of trans-resveratrol against the cytotoxicity of chloroacetanilide herbicides in vitro.

The beneficial effect of trans-resveratrol (RESV) on health is well documented. Our aim was to study the putative preventive effect of RESV on the cytotoxicity of frequently used herbicides (alachlor, acetochlor). Estrogen receptor positive (ER+) MCF-7 human mammary carcinoma, HepG2 (ER+) human hepatocellular carcinoma and VERO estrogen receptor negative (ER-) non-transformed monkey fibroblast cell lines were treated with alachlor and acetochlor (2-500 microg/ml) as toxic agents, and RESV (10 microM) as preventive agent. The MTT dye reduction assay was performed to test cytotoxicity, and flow cytometry to test cell proliferation and apoptosis. RESV is not cytotoxic in the concentration range of 1-100 microM on neither cell lines examined after 24 h, but cytotoxic on Vero and MCF-7 cells at 100 microM after 48h, and on all three cell lines after 72 h. On both ER+ cell lines a stimulation of viability occurs in the low concentration range (0.5-12.5 microM) as detected by the MTT assay. Cell cycle analysis of the culture shows a significant increase of S-phase cells at low concentrations of RESV (10-50 microM) and a decrease in the 100-200 microM concentration range. The ratio of apoptotic cells significantly increases after the administration of 50 microM RESV, depending on the incubation time. The cytotoxicity of 20-65 microg/ml alachlor and 10-65 microg/ml acetochlor was significantly decreased by the addition of 10 microM RESV in Vero ER- cells whereas no significant change was detected on ER+ cell lines MCF-7 and HepG2. These results show that RESV protects non-transformed ER- cells, but has no such effect on ER+ tumor cells.

Lafutidine-induced increase in intracellular ca(2+) concentrations in PC12 and endothelial cells.

Lafutidine, a histamine H(2) receptor antagonist, exerts gastroprotective effects in addition to gastric antisecretory activity. The gastrointestinal protective effects of lafutidine are mediated by capsaicin-sensitive neurons, where capsaicin excites neurons by opening a member of the transient receptor potential channel family (TRPV1). Since the effect of lafutidine on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in cells has not been elucidated, we investigated the lafutidine response to [Ca(2+)](i) in rat pheochromocytoma PC12 and human endothelial cells. Lafutidine at pharmacological concentrations greater than 1 mM induced a sustained increase in [Ca(2+)](i) in the presence of extracellular CaCl(2) in PC12 cells, while capsaicin showed dual effects on [Ca(2+)](i) in PC12 cells, where it activated TRPV1 and inhibited store-operated Ca(2+) entry. The thapsigargin (an activator of store-operated Ca(2+) entry)-induced increase in [Ca(2+)](i) in PC12 cells was inhibited by capsaicin and SKF96365, an inhibitor of store-operated Ca(2+) entry, and the lafutidine response was inhibited by capsaicin but not by SKF96365. In endothelial cells, lafutidine induced an increase in [Ca(2+)](i) in a SKF96365-insensitive manner. These results suggest that lafutidine stimulates Ca(2+) entry via the capsaicin-sensitive pathway but not the SKF96365-sensitive pathway. The possible role of store-operated Ca(2+) entry induced by lafutidine on gastrointestinal function is also discussed.

Inhibition of zaleplon metabolism by cimetidine in the human liver: in vitro studies with subcellular fractions and precision-cut liver slices.

1. The effect of cimetidine on the metabolism of zaleplon (ZAL) in human liver subcellular fractions and precision-cut liver slices was investigated. 2. ZAL was metabolized to a number of products including 5-oxo-ZAL (M2), which is known to be formed by aldehyde oxidase, N-desethyl-ZAL (DZAL), which is known to be formed by CYP3A forms, and N-desethyl-5-oxo-ZAL (M1). 3. Human liver microsomes catalysed the NADPH-dependent metabolism of ZAL to DZAL. Kinetic analysis of three microsomal preparations revealed mean (+/-SEM) S(50) and V(max) of 310 +/- 24 micro M and 920 +/- 274 pmol/min/mg protein, respectively. 4. Human liver cytosol preparations catalysed the metabolism of ZAL to M2. Kinetic analysis of three cytosol preparations revealed mean (+/-SEM), K(m) and V(max) of 124 +/- 14 micro M and 564 +/- 143 pmol/min/mg protein, respectively. 5. Cimetidine inhibited ZAL metabolism to DZAL in liver microsomes and to M2 in the liver cytosol. With a ZAL substrate concentration of 62 micro M, the calculated mean (+/-SEM, n = 3) IC50 were 596 +/- 103 and 231 +/- 23 micro M for DZAL and M2 formation, respectively. Kinetic analysis revealed that cimetidine was a competitive inhibitor of M2 formation in liver cytosol with a mean (+/-SEM, n = 3) K(i) of 155 +/- 16 micro M. 6. Freshly cut human liver slices metabolized ZAL to a number of products including 1, M2 and DZAL. 7. Cimetidine inhibited ZAL metabolism in liver slices to M1 and M2, but not to DZAL. Kinetic analysis revealed that cimetidine was a competitive inhibitor of M2 formation in liver slices with an average (n = 2 preparations) K(i) of 506 micro M. 8. The results demonstrate that cimetidine can inhibit both the CYP3A and aldehyde oxidase pathways of ZAL metabolism in the human liver. Cimetidine appears to be a more potent inhibitor of aldehyde oxidase than of CYP3A forms and hence in vivo is likely to have a more marked effect on ZAL metabolism to M2 than on DZAL formation. 9. The results also demonstrate that precision-cut liver slices may be a useful model system for in vitro drug-interaction studies.

Effect of a novel NSAID derivative with antioxidant moiety on oxidative damage caused by liver and cerebral ischaemia-reperfusion in rats.

Tissue ischaemia-reperfusion evokes toxic and harmful biochemical processes such as oxidative stress and inflammation. The aim of this study is to investigate the indices of tissue damage in rat liver and brain after ischaemia-reperfusion injury of these organs, and to study prospective cytoprotection of molecules such as the novel anti-inflammatory N-(2-thiolethyl)-2-(2-[N'-(2,6-dichlorophenyl)amino] phenyl)acetamide (compound 1) and alpha-tocopherol. Two experimental models were studied: firstly, 30 min liver ischaemia via hepatoduodenal ligament clamping followed by 60 min reperfusion; and secondly, 45 min cerebral ischaemia via bilateral common carotid artery occlusion followed by 90 min reperfusion. Compound 1 and alpha-tocopherol were administered intraperitoneally before induction of ischaemia. We hereby report that compound 1, a molecule that combines potent in-vitro antioxidant and in-vivo anti-inflammatory activity with low gastrointestinal toxicity, offered protection in-vivo against liver or brain ischaemia-reperfusion-induced damage. Both compound 1 and alpha-tocopherol prevented changes in lipid peroxidation in the rat liver and brain tissue and in tumour necrosis factor (TNF-alpha) levels in brain. Also compound 1 attenuated glutathione depletion, evoked by ischaemia-reperfusion, in the rat brain but not in the liver. These results could be explained on the basis of the antioxidant/anti-inflammatory properties of compound 1 and suggest its beneficial effect and potential therapeutic use in post-ischaemic injury.

Activin A inhibits growth and hexamethylene bisacetamide-induced differentiation in human retinoblastoma Y-79 cells.

Activin regulates the growth and differentiation of a variety of cells and is a member of the transforming growth factor-beta (TGF-beta) family. Previously, we found that the retinoblastoma cell line Y-79 expresses both activins and activin receptors, suggesting that activin may have an autocrine function in these cells. In this study, the effects of exogeneous activin A on cultured Y-79 cells were examined. The results demonstrate that activin A inhibits hexamethylene bisacetamide (HMBA) -induced Y-79 cell differentiation in both serum-containing and serum-free medium. Activin A also inhibits Y-79 cell growth in serum-containing medium but not in serum-free medium.

A pharmacological profile of the novel, peripherally-selective kappa-opioid receptor agonist, EMD 61753.

1. The pharmacological properties of the novel diarylacetamide kappa-opioid receptor agonist, EMD 61753, have been compared with those of ICI 197067 (a centrally-acting kappa agonist) and ICI 204448 (a peripherally-selective kappa agonist). 2. EMD 61753 binds with high affinity (IC50 5.6 nM) and selectivity (kappa:mu:delta:sigma binding ratio 1:536:125: > 1,786) to kappa-opioid receptors and is a full and potent (IC50 54.5 nM) agonist in an in vitro assay for kappa-opioid receptors (rabbit vas deferens preparation). 3. Systemically-applied [14C]-EMD 61753 is found in high concentrations in the lungs, liver, adrenal glands and kidneys. Considerably less radioactivity is detected in the whole brain, and this radioactivity is concentrated in the region of the cerebral ventricles in the choroid plexuses. EMD 61753 penetrates only poorly into the CNS. 4. EMD 61753 was weakly effective in pharmacological tests of central activity. This compound reversed haloperidolol-induced DOPA accumulation in the nucleus accumbens of the rat only at a dose of 30 mg kg-1, s.c., (doses of 0.1, 1.0 and 10 mg kg-1, s.c., and 1.0, 10 and 100 mg kg-1, p.o., were inactive). Hexobarbitone-induced sleeping in mice was prolonged by EMD 61753 at threshold doses of 10 mg kg-1, s.c., and 100 mg kg-1, p.o., whereas the motor performance of rats in the rotarod test was impaired by EMD 61753 with an ID50 value of 453 mg kg-1, s.c. 5. EMD 61753 produced dose-dependent, naloxone-reversible antinociception in the mouse formalin test (1st phase ID50 1.9 mg kg-1, s.c., and 10.4 mg kg-1, p.o.; 2nd phase ID50 0.26 mg kg-1, s.c., and 3.5 mg kg-1, p.o.) and rodent abdominal constriction test (ID50 mouse 1.75 mg kg-1, s.c., and 8.4 mg kg-1, p.o.; ID50 rat 3.2 mg kg-1, s.c., and 250 mg kg-1, p.o.). EMD 61753 was inactive, or only weakly effective, in the rat pressure test under normalgesic conditions. After the induction of hyperalgesia with carrageenin, however, this compound elicited potent, dose-dependent (ID50 0.08 mg kg-1, s.c., and 6.9 mg kg-1, p.o., after remedial application, and 0.2 mg kg-1, s.c., and 3.1 mg kg-1, p.o., after prophylactic application) and naloxone-reversible antinociception. The antinociceptive action of systemically-applied (50 mg kg-1, p.o.) EMD 61753 in the hyperalgesic pressure test was completely inhibited by injection of the K-opioid antagonist norbinaltorphimine (100 Lg) into the inflamed tissue, a result which indicates that this opioid effect is mediated peripherally.6. Cutaneous plasma protein extravasation produced by antidromic electrical stimulation of the rat saphenous nerve was dose-dependently inhibited by systemically-applied EMD 61753 (ID13 values 3.7 mg kg-1, s.c., and 35.8 mg kg-1, p.o.), and this effect was completely antagonized by intraplantar application of norbinaltorphimine (50 microg). Extravasation elicited by the intraplantar application of substance P (10 microg) was not influenced by the administration of EMD 61753.7. EMD 61753 produced dose-dependent diuresis in non-hydrated rats at doses of and above 1.0 mg kg-1, s.c., and 10 mg kg-1, p.o., and in saline-loaded rats at doses of and above 10 mg kg-1, s.c.,and 30mgkg-1, p.o.8. The prostaglandin-mediated fall in mean arterial blood pressure elicited in anaesthetized rats by i.v.application of arachidonic acid was not inhibited by prior treatment with EMD 61753 (10mg kg-1,p.o.). Thus, a blockade of prostaglandin synthesis via inhibition of cyclo-oxygenase activity does not contribute to the in vivo effects of EMD 61753 and its metabolites.9 The present experiments therefore indicate that EMD 61753 is a potent, selective and orally-effective full ic-opioid receptor agonist which has a limited ability to penetrate the blood-brain barrier and elicit centrally-mediated sedation, putative aversion, diuresis, and antinociception. The inhibitory actions of systemically-applied EMD 61753 against hyperalgesic pressure nociception and neurogenic inflammation are mediated peripherally, probably by opioid receptors on the endings of sensory nerve fibres.

Kappa opioid antagonist effects of systemically administered nor-binaltorphimine in a thermal antinociception assay in rhesus monkeys.

The effects of subcutaneously administered nor-binaltorphimine (nor-BNI; 1.0 and 3.2 mg/kg) were examined in the warm-water (50 degrees C and 55 degrees C) tail-withdrawal assay in rhesus monkeys (n = 3). Nor-BNI alone produced variable antinociceptive effects in 50 degrees C water up to 3.5 hr after administration but was completely ineffective against the 55 degrees C stimulus. Pretreatment with nor-BNI under conditions where it was devoid of antinociceptive effects produced rightward shifts in dose-effect curves for the kappa opioid agonist U50,488 for as long as 14 and 21 days after 1.0 and 3.2 mg/kg of nor-BNI, respectively. Under conditions when U50,488 dose-effect curves were shifted, nor-BNI (3.2 mg/kg) also caused rightward shifts in the antinociceptive dose-effect curves of the kappa agonist U69,593 but not in those of the mu agonist alfentanil or the kappa agonists [5R-(5,7,8,beta)]N-methyl-N-[7- (1-pirrolidinyl)1-oxaspiro[4,5]dec-8-yl]4-benzofuranaceta mide, bremazocine, ethylketocyclazocine and Mr2033. It is concluded that under the present conditions, nor-BNI acts as a selective kappa opioid antagonist with an extremely long duration of action. These findings are also consistent with the notion that nor-BNI may antagonize only compounds acting at a subtype of kappa opioid receptor.

Acute oral toxicity of insect repellent N,N-diethylphenylacetamide in mice, rats and rabbits and protective effect of sodium pentobarbital.

Median lethal dose (LD50) of undiluted liquid insect repellent N,N-diethylphenylacetamide (DEPA) in male mice, rats and rabbits was 900, 825 and 635 mg/kg respectively when administered by gavage. Signs of DEPA intoxication point to stimulation of central nervous system (CNS). Acetazolamide (10 mg/kg), sodium bicarbonate (40 mg/kg), and atropine (5 mg/kg) when injected (ip) 5 min after a lethal oral dose of DEPA (1700 mg/kg) did not prevent mortality, while sodium pentobarbital (SPB; 20 mg/kg) when injected 5 min after or 15 min before DEPA provided greater protection to the animals. SPB pretreatment elevated the LD50 of DEPA to 1780 and 1535 mg/kg in mice and rats respectively and 85% rats survived when SPB was injected 5 min after acute oral exposure to DEPA (1000 mg/kg). Carboxylesterase (CaE) inhibition is not a factor in the protection mechanism of SPB. DEPA (1000 mg/kg) when given orally elevated blood PCO2 and reduced pH, O2 content and per cent O2 saturation, while administration of SPB after the same dose of DEPA reduced the degree of acidosis and raised PCO2, and increased the O2 content and per cent O2 saturation to near normal status. The CNS depressant action of SPB may be a crucial factor in protection of rats from DEPA poisoning.

Expression and phosphorylation of the retinoblastoma protein during induced differentiation of murine erythroleukemia cells.

Phosphorylation and dephosphorylation of the retinoblastoma protein, pRB, play a role in the control of cell cycle progression and expression of differentiation in eukaryotic cells. The regulation of pRB level and phosphorylation state was investigated during the induction of differentiation of murine erythroleukemia cells (MELC) by the chemical agent hexamethylene bisacetamide (HMBA). In MELC, there is a critical time in G1 or early S phase when HMBA must be present in order to induce differentiation. This is followed by prolongation of the subsequent G1 phase, resumption of progression through the cell cycle for several generations, and then cell cycle arrest in G1-G0. Associated with HMBA-induced prolongation of G1, there is an increase in the amount of the underphosphorylated form of pRB. A variant cell line (DS19/VCR-C) with accelerated kinetics of HMBA-mediated differentiation shows a more marked increase in underphosphorylated pRB. In culture with HMBA, as MELC resume progression through the cell cycle, pRB is present in the phosphorylated form. The total amount of pRB increases approximately 3-fold over the succeeding cell divisions prior to terminal arrest in G1. This increase in pRB is inhibited by dexamethasone, which also blocks HMBA-induced MELC differentiation. During this period, RB mRNA also increases approximately 3- to 5-fold, which reflects an increase in the rate of transcription, with no change in mRNA stability. The state of phosphorylation and amount of pRB appear to be involved in the control of HMBA-induced terminal cell division of MELC.