Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation.

The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5 hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5 mC) and 5 hmC at a CCGG site within the 5' γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5 mC and 5 hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5 hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5 hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5 hmC and negatively correlated with 5 mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5 hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter.

RN-1, a potent and selective lysine-specific demethylase 1 inhibitor, increases γ-globin expression, F reticulocytes, and F cells in a sickle cell disease mouse model.

Increased levels of fetal hemoglobin are associated with decreased symptoms and increased lifespan in patients with sickle cell disease (SCD). Hydroxyurea, the only drug currently approved for SCD, is not effective in a large fraction of patients, and therefore, new agents are urgently needed. Recently it was found that lysine demethylase 1, an enzyme that removes monomethyl and dimethyl residues from the lysine 4 residue of histone H3, is a repressor of γ-globin gene expression. In this article, we have compared the ability of tranylcypromine (TCP) and a more potent TCP derivative, RN-1, to increase γ-globin expression in cultured baboon erythroid progenitor cells and in the SCD mouse model. The results indicate that the ability of RN-1 to induce F cells and γ-globin mRNA in SCD mice is similar to that of decitabine, the most powerful fetal hemoglobin-inducing drug known, and greater than that of either TCP or hydroxyurea. We conclude that RN-1 and other lysine demethylase 1 inhibitors may be promising new γ-globin-inducing agents for the treatment of SCD that warrant further studies in other preclinical models, such as nonhuman primates.

Rines E3 ubiquitin ligase regulates MAO-A levels and emotional responses.

Monoamine oxidase A (MAO-A), the catabolic enzyme of norepinephrine and serotonin, plays a critical role in emotional and social behavior. However, the control and impact of endogenous MAO-A levels in the brain remains unknown. Here we show that the RING finger-type E3 ubiquitin ligase Rines/RNF180 regulates brain MAO-A subset, monoamine levels, and emotional behavior. Rines interacted with MAO-A and promoted its ubiquitination and degradation. Rines knock-out mice displayed impaired stress responses, enhanced anxiety, and affiliative behavior. Norepinephrine and serotonin levels were altered in the locus ceruleus, prefrontal cortex, and amygdala in either stressed or resting conditions, and MAO-A enzymatic activity was enhanced in the locus ceruleus in Rines knock-out mice. Treatment of Rines knock-out mice with MAO inhibitors showed genotype-specific effects on some of the abnormal affective behaviors. These results indicated that the control of emotional behavior by Rines is partly due to the regulation of MAO-A levels. These findings verify that Rines is a critical regulator of the monoaminergic system and emotional behavior and identify a promising candidate drug target for treating diseases associated with emotion.

Mechanism-based inhibition of cytochrome P450 (CYP)2A6 by chalepensin in recombinant systems, in human liver microsomes and in mice in vivo.

BACKGROUND AND PURPOSE: Chalepensin is a pharmacologically active furanocoumarin compound found in rue, a medicinal herb. Here we have investigated the inhibitory effects of chalepensin on cytochrome P450 (CYP) 2A6 in vitro and in vivo. EXPERIMENTAL APPROACH: Mechanism-based inhibition was studied in vitro using human liver microsomes and bacterial membranes expressing genetic variants of human CYP2A6. Effects in vivo were studied in C57BL/6J mice. CYP2A6 activity was assayed as coumarin 7-hydroxylation (CH) using HPLC and fluorescence measurements. Metabolism of chalepensin was assessed with liquid chromatography/mass spectrometry (LC/MS). KEY RESULTS: CYP2A6.1, without pre-incubation with NADPH, was competitively inhibited by chalepensin. After pre-incubation with NADPH, inhibition by chalepensin was increased (IC(50) value decreased by 98%). This time-dependent inactivation (k(inact) 0.044 min(-1) ; K(I) 2.64 µM) caused the loss of spectrally detectable P450 content and was diminished by known inhibitors of CYP2A6, pilocarpine or tranylcypromine, and by glutathione conjugation. LC/MS analysis of chalepensin metabolites suggested an unstable epoxide intermediate was formed, identified as the corresponding dihydrodiol, which was then conjugated with glutathione. Compared with the wild-type CYP2A6.1, the isoforms CYP2A6.7 and CYP2A6.10 were less inhibited. In mouse liver microsomes, pre-incubation enhanced inhibition of CH activity. Oral administration of chalepensin to mice reduced hepatic CH activity ex vivo. CONCLUSIONS AND IMPLICATIONS: Chalepensin was a substrate and a mechanism-based inhibitor of human CYP2A6. Formation of an epoxide could be a key step in this inactivation. 'Poor metabolizers' carrying CYP2A6*7 or *10 may be less susceptible to inhibition by chalepensin. Given in vivo, chalepensin decreased CYP2A activity in mice.

Simple, direct, and informative method for the assessment of CYP2C19 enzyme inactivation kinetics.

Many clinically relevant drug interactions involving cytochrome P450 inhibition are mediated by mechanism-based inactivation (MBI). Time-dependent inhibition is one of the major features distinguishing between reversible inhibition and MBI. It thus provides a useful screening approach for early drug interaction risk assessment. Accordingly, we developed an easy and informative fluorometric method for the assessment of CYP2C19 enzyme inactivation kinetics. Dibenzylfluorescein (DBF) is widely used as a profluorescent probe substrate for P450 activity and inhibition assays, but its use has been considered to be limited to traditional endpoint assays. We monitored CYP2C19-catalyzed metabolism of DBF using synthesized fluorescein benzyl ester and fluorescein benzyl ether along with commercially available fluorescein as intermediate standards. Furthermore, we demonstrated the use of DBF in a kinetic assay as a progress curve analysis for straightforward determination of whether a compound is a time-dependent inactivator of CYP2C19. The recombinant human CYP2C19 inactivation kinetics of isoniazid, ticlopidine, and tranylcypromine were evaluated, and their key kinetic parameters were measured from the same experiment. The known mechanism-based inactivators, isoniazid and ticlopidine, exhibited clear time-dependent inactivation with K(I) and k(inact) values of 250.5 ± 34 µM and 0.137 ± 0.006 min(-1) and 1.96 ± 0.5 µM and 0.135 ± 0.009 min(-1), respectively. Tranylcypromine did not display any time-dependent inhibition, which is consistent with its reported mechanism of competitive inhibition. In summary, DBF is suitable for use in the progress curve analysis approach and can be used as an initial screen to identify compounds that require more detailed investigations in drug interaction optimization.

Chronic ouabain treatment increases the contribution of nitric oxide to endothelium-dependent relaxation.

The aim of this study was to analyze the contribution of nitric oxide, prostacyclin and endothelium-dependent hyperpolarizing factor to endothelium-dependent vasodilation induced by acetylcholine in rat aorta from control and ouabain-induced hypertensive rats. Preincubation with the nitric oxide synthase inhibitor N-omega-nitro-L-arginine methyl esther (L-NAME) inhibited the vasodilator response to acetylcholine in segments from both groups but to a greater extent in segments from ouabain-treated rats. Basal and acetylcholine-induced nitric oxide release were higher in segments from ouabain-treated rats. Preincubation with the prostacyclin synthesis inhibitor tranylcypromine or with the cyclooxygenase inhibitor indomethacin inhibited the vasodilator response to acetylcholine in aortic segments from both groups. The Ca2+-dependent potassium channel blocker charybdotoxin inhibited the vasodilator response to acetylcholine only in segments from control rats. These results indicate that hypertension induced by chronic ouabain treatment is accompanied by increased endothelial nitric oxide participation and impaired endothelium-dependent hyperpolarizing factor contribution in acetylcholine-induced relaxation. These effects might explain the lack of effect of ouabain treatment on acetylcholine responses in rat aorta.

Chronic ouabain treatment increases the contribution of nitric oxide to endotheliumdependent relaxation

The aim of this study was to analyze the contribution of nitric oxide, prostacyclinand endothelium-dependent hyperpolarizing factor to endothelium-dependentvasodilation induced by acetylcholine in rat aorta from control and ouabain-inducedhypertensive rats. Preincubation with the nitric oxide synthase inhibitor N-omeganitro-L-arginine methyl esther (L-NAME) inhibited the vasodilator response toacetylcholine in segments from both groups but to a greater extent in segments fromouabain-treated rats. Basal and acetylcholine-induced nitric oxide release were higherin segments from ouabain-treated rats. Preincubation with the prostacyclin synthesisinhibitor tranylcypromine or with the cyclooxygenase inhibitor indomethacininhibited the vasodilator response to acetylcholine in aortic segments from bothgroups. The Ca2+-dependent potassium channel blocker charybdotoxin inhibited thevasodilator response to acetylcholine only in segments from control rats. Theseresults indicate that hypertension induced by chronic ouabain treatment is accompaniedby increased endothelial nitric oxide participation and impaired endotheliumdependenthyperpolarizing factor contribution in acetylcholine-induced relaxation.These effects might explain the lack of effect of ouabain treatment on acetylcholineresponses in rat aorta (AU)

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The guinea pig forced swim test as a new behavioral despair model to characterize potential antidepressants.

RATIONALE: Behavioral despair is a model of high predictivity for antidepressant activity in murids. For some drug targets, guinea pigs exhibit a higher homology to their human counterparts compared to murids. OBJECTIVES: In this paper, we established a model of behavioral despair namely, the forced swim test (FST) in guinea pigs. MATERIALS AND METHODS: Male guinea pigs underwent the FST similar to rats. Animals received intraperitoneal injections of either vehicle or drugs 24, 4, and 0.5 h before testing. We tested the tricyclic antidepressants desipramine and amitriptyline, the monoamine oxidase inhibitor tranylcypromine, the selective serotonin reuptake inhibitors fluoxetine and paroxetine, and the neurokinin 1 (NK(1)) receptor antagonist, L-733,060, and for comparison the antipsychotic clozapine and the stimulant methamphetamine. RESULTS: Desipramine (> or =3 mg/kg) and amitriptyline (>10 mg/kg) increased the latency to immobility (LTI) to greater than 230 s, and tranylcypromine (10 mg/kg) it to greater than 190 s. Paroxetine (>0.3 mg/kg) and fluoxetine (>10 mg/kg) also increased LTI significantly but only to greater than 120 s. Methamphetamine (3 mg/kg) completely eliminated immobility, whereas clozapine (5-20 mg/kg) had no effect. L-733,060 (10 mg/kg) increased LTI to 270 s. Doses producing significant effects in FST were investigated in the open field. Antidepressants did not affect locomotion, whereas methamphetamine induced hyperlocomotion. CONCLUSIONS: We demonstrate the suitability of a modified procedure of the FST for a nonmurid species: the guinea pig. Known antidepressants showed similar effects as in rats and mice. It is interesting to note that the NK(1) antagonist L-733,060 increased forced swimming, suggesting its antidepressant potential. Thus, the guinea pig FST allows the study of antidepressant activity also in NK(1) antagonists that cannot be studied appropriately in murids.

Honokiol up-regulates prostacyclin synthease protein expression and inhibits endothelial cell apoptosis.

Honokiol is a bioactive compound extracted from the Chinese medicinal herb Magnolia officinalis. We recently demonstrated that honokiol inhibited arterial thrombosis through stimulation of prostacyclin (PGI2) generation and endothelial cell protection. The current study is designed to investigate its mechanism of stimulation of PGI2 generation and cell protection. 6-keto-PGF1alpha, the stable metabolite of PGI2, in the media of rat aortic endothelial cells was measured with radioimmunoassay kits. Indomethacin, an inhibitor of cyclooxygenase (COX) and tranylcypromine, a prostacyclin synthease inhibitor were used to ascertain the target enzyme affected by honokiol. Prostacyclin synthease protein levels in endothelial cells were determined by Western blot analysis using an anti-PGI2 synthease rabbit polyclonal antibody. Flow cytometry was used to quantify the apoptotic cells and spectrophotometry was used to test the caspase-3 activity. Honokiol (0.376-37.6 microM) increased the level of 6-keto-PGF1alpha in the media of normal endothelial cells. It counteracted the inhibitory effect of tranylcypromine on the PGI2 generation, but did not influence the effect of indomethacin; evidently, honokiol up-regulated the expression of prostacyclin synthease in the endothelial cells. These effects showed perfect concentration-dependent behavior. In addition, at lower concentration (0.376-3.76 microM), honokiol significantly decreased the percentage of apoptotic endothelial cells induced by oxidized low-density lipoprotein (ox-LDL) and significantly lowered the activity of caspase-3 stimulated by ox-LDL. A high dose of honokiol (37.6 microM), however, failed to influence either of them. In conclusion, honokiol augments PGI2 generation in normal endothelial cells; its effect on PGI2 generation attributes to up-regulation of prostacyclin synthease expression; its cell protection may be correlated with its inhibition on apoptosis of endothelial cells. These findings have partly revealed the molecular mechanism of honokiol on inhibiting arterial thrombosis.

Nitric oxide synthesis is required for exercise-induced increases in hippocampal BDNF and phosphatidylinositol 3' kinase expression.

Previous studies have shown that running exercise, either alone or in combination with antidepressant treatment, results in increased hippocampal BDNF levels. Nitric oxide (NO) is an important signaling molecule that has neuronal survival-promoting properties and has been shown to play an important role in plasticity associated with activating interventions. Herein, we administered the NO synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (L-NAME), in conjunction with the monoamine oxidase inhibitor (MAOI) antidepressant, tranylcypromine, and voluntary wheel-running exercise to determine whether the enhancement in full-length BDNF mRNA occurring with these interventions is dependent upon NO synthesis. Our results demonstrate that both chronic exercise and chronic exercise-plus-tranylcypromine lead to enhanced hippocampal BDNF mRNA and protein expression. NOS inhibition prevents this effect of chronic exercise, but only partly prevents the effects of the exercise/antidepressant combination. Thus, the robust enhancement in BDNF mRNA occurring with exercise appears to be NO synthesis-dependent, but the intervention including antidepressant may enhance BDNF expression through alternative intracellular mechanisms. In addition, because exercise and antidepressants have both been shown to activate survival-promoting genes, we evaluated the levels of hippocampal phosphatidylinositol 3' kinase (PI-3K), an important signaling molecule within a principal neuronal survival-promoting intracellular pathway. Like BDNF mRNA and protein, exercise increases the expression of PI-3K, whereas concomitant NOS inhibition prevents this increase in PI-3K immunoreactivity above control levels. Our results are discussed in light of possible overlapping, but distinct intracellular pathways activated by exercise and antidepressant treatment to bring about enhancements in BDNF expression and other survival-promoting effects. These findings further demonstrate the potential therapeutic potential of chronic exercise to supplement pharmacotherapeutic treatment of mood disorders.

Enhancement of drug affinity for cell membranes by conjugation with lipoamino acids. I. Synthesis and biological evaluation of lipophilic conjugates of tranylcypromine.

Conjugation with lipoamino acids (LAAs) increases the lipophilicity of drug molecules. Because of their amphipatic nature, they also provide the conjugated drugs a 'membrane-like character', capable to facilitate their interaction with and penetration through cell membranes and biological barriers. To study such a feature, our aim is to collect experimental and computational data using a novel series of lipophilic conjugates between a model drug (tranylcypromine (TCP)) and LAA residues containing a short, a medium or a long alkyl side chain (C-4 to C-16), to provide a wide range of lipophilicity. For comparison, a corresponding set of amides of TCP with alkanoic or fatty acids was prepared and characterized. Their in vitro monoamine oxidase inhibitory activity also tested.

Synthesis and antidepressant activities of some 3,5-diphenyl-2-pyrazolines.

Ten new 3,5-diphenyl-2-pyrazoline derivatives were synthesised by reacting 1,3-diphenyl-2-propen-1-one with hydrazine hydrate. The chemical structures of the compounds were proved by means of their IR, 1H-NMR spectroscopic data and microanalyses. The antidepressant activities of these compounds were evaluated by the 'Porsolt Behavioural Despair Test' on Swiss-Webster mice. 3-(4-Methoxyphenyl)-5-(3,4-dimethoxyphenyl)-2-pyrazoline, 3-(4-methoxyphenyl)-5-(2-chloro-3,4-dimethoxyphenyl)-2-pyrazoline and 3-(4-chlorophenyl)-5-(2-chloro-3,4-dimethoxyphenyl)-2-pyrazoline reduced 41.94-48.62% immobility times at 100 mgkg(-1) dose level. In addition, it was found that 4-methoxy and 4-chloro substituents on the phenyl ring at position 3 of the pyrazoline ring increased the antidepressant activity; the replacement of these groups by bromo and methyl substituents decreased activity in mice.

Evaluation of methoxsalen, tranylcypromine, and tryptamine as specific and selective CYP2A6 inhibitors in vitro.

CYP2A6 is the principle enzyme metabolizing nicotine to its inactive metabolite cotinine. In this study, the selective probe reactions for each major cytochrome P450 (P450) were used to evaluate the specificity and selectivity of the CYP2A6 inhibitors methoxsalen, tranylcypromine, and tryptamine in cDNA-expressing and human liver microsomes. Phenacetin O-deethylation (CYP1A2), coumarin 7-hydroxylation (CYP2A6), diclofenac 4'-hydroxylation (CYP2C9), omeprazole 5-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), 7-ethoxy-4-trifluoromethylcoumarin deethylation (CYP2B6), p-nitrophenol hydroxylation (CYP2E1), and omeprazole sulfonation (CYP3A4) were used as index reactions. Apparent K(i) values for inhibition of P450s' (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) activities showed that tranylcypromine, methoxsalen, and tryptamine have high specificity and relative selectivity for CYP2A6. In cDNA-expressing microsomes, tranylcypromine inhibited CYP2A6 (K(i) = 0.08 microM) with about 60- to 5000-fold greater potency relative to other P450s. Methoxsalen inhibited CYP2A6 (K(i) = 0.8 microM) with about 3.5- 94-fold greater potency than other P450s, except for CYP1A2 (K(i) = 0.2 microM). Tryptamine inhibited CYP2A6 (K(i) = 1.7 microM) with about 6.5- 213-fold greater potency relative to other P450s, except for CYP1A2 (K(i) = 1.7 microM). Similar results were also obtained with methoxsalen and tranylcypromine in human liver microsomes. R-(+)-Tranylcypromine, (+/-)-tranylcypromine, and S-(-)-tranylcypromine competitively inhibited CYP2A6-mediated metabolism of nicotine with apparent K(i) values of 0.05, 0.08, and 2.0 microM, respectively. Tranylcypromine [particularly R-(+) isomer], tryptamine, and methoxsalen are specific and relatively selective for CYP2A6 and may be useful in vivo to decrease smoking by inhibiting nicotine metabolism with a low risk of metabolic drug interactions.

Regulation of c-Fos and NGF1-A by antidepressant treatments.

The influence of antidepressant treatments on the expression of c-Fos and NGF-1A, two immediate early gene (IEG) transcription factors, was examined. Administration of electroconvulsive seizures (ECS), tranylcypromine, or imipramine, three different classes of antidepressants, increased the expression of c-Fos mRNA and immunoreactivity in rat frontal cortex, but the magnitude of the increase for each treatment differed and the effect of imipramine was preceded by inhibition of c-Fos expression. Expression of NGF-1A was increased by acute or chronic administration of ECS or tranylcypromine, and by chronic (21 d), but not acute, administration of imipramine. To study the mechanisms underlying these differences, we examined the neurotransmitter receptors that regulate the expression of c-Fos. ECS- and tranylcypromine induction of c-Fos immunoreactivity in frontal cortex was partially inhibited by pretreatment with specific antagonists for alpha 1-adrenergic, beta-adrenergic, and 5-HT2A/2C, but not D2-dopamine receptors. ECS induction of c-Fos was also inhibited by D1-dopamine and NMDA glutamate receptor antagonists, suggesting that the greater induction of c-Fos by ECS results from activation of these, and possibly other, neurotransmitter receptors. In the hippocampus, antagonism of tranylcypromine was similar to that in frontal cortex, except the D1-dopamine receptor antagonist also blocked the c-Fos response. In contrast, antagonism of the ECS response in hippocampus was only blocked by the NMDA receptor antagonist. The results demonstrate that ECS- and tranylcypromine induction of c-Fos is mediated by activation of several different neurotransmitter receptors, but that the exact pharmacological profile is different for each treatment and brain region.

Studies on the synthesis and antidepressant activity of some 1-thiocarbamoyl-3,5-diphenyl-2-pyrazolines.

Several 1-thiocarbamoyl-3,5-diphenyl-2-pyrazoline derivatives have been synthesized by reacting 1,3-diphenyl-2-propen-1-one (chalcones) with thiosemicarbazide, or hydrazine hydrate and then with various isothiocyanates. Their chemical structures have been proven by UV, IR, 1H-NMR and elemental analysis. Antidepressant activities were investigated by "Porsolt's Behavioural Despair Test". 1-Thiocarbamoyl-3-(phenyl and 4-chlorophenyl-5-(4-methyl and 4-methoxyphenyl-2-pyrazoline (compounds III, IV, VII, VIII) and 3-(4-methylphenyl)-5-(4-methoxyphenyl)-2-pyrazoline (compound XII) showed equivalent or higher activity than pargyline hydrochloride and tranylcypromine sulfate.

Effects of the antidepressant/antipanic drug phenelzine on GABA concentrations and GABA-transaminase activity in rat brain.

The effects of long-term (28-day) administration of several antidepressant/antipanic drugs [imipramine, desipramine, tranylcypromine and phenelzine (PLZ)] on gamma-aminobutyric acid-tranaminase (GABA-T) activity and GABA levels were investigated in rat frontal cortex. Of the drugs investigated, only PLZ inhibited GABA-T and elevated GABA levels. Additional short-term experiments were conducted with PLZ, and they demonstrated a dose-dependent inhibition of GABA-T in rat whole brain. Time-response studies on inhibition of GABA-T in whole brain demonstrated that at a dose of PLZ of 15 mg/kg i.p. inhibition of GABA-T remained relatively constant from 1 to 8 hr and that the enzyme was still inhibited by 23% at 24 hr after PLZ administration.

The effects of Ca2+ antagonists and hydralazine on central 5-hydroxytryptamine biochemistry and function in rats and mice.

1. The effects of calcium antagonists on behaviour mediated by 5-hydroxytryptamine (5-HT) have been studied in rats and mice together with an investigation of the effects of these drugs on 5-HT synthesis in rat brain and endogenous 5-HT release from brain slices. 2. Administration of felodipine (35 mg kg-1 i.p.) to rats pretreated with tranylcypromine (20 mg kg-1, i.p.) resulted in the animals displaying the complete 5-HT-mediated behavioural syndrome (including head weaving, reciprocal forepaw treading and hind limb abduction) 75 min later. No evidence was obtained for the rate of 5-HT synthesis in brain regions differing between control and felodipine-treated rats. 3. Pretreatment with felodipine (10 or 35 mg kg-1) enhanced the 5-HT-mediated behavioural syndrome induced by injection of tranylcypromine and L-tryptophan. The rate of 5-HT accumulation in the brain was similar in both groups. Administration of Bay K 8644 (1 mg kg-1, i.p.) did not prevent the enhanced behaviour induced by felodipine (10 mg kg-1). 4. The 5-HT behavioural syndrome induced by injection of the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was unaltered by either acute injection of felodipine (35 mg kg-1) or administration of felodipine twice daily for 3 days. 5. Felodipine (10 microM), verapamil (10 microM) and Bay K 8644 (10 microM) did not alter either basal release of endogenous 5-HT from slices prepared from frontal cortex or hind brain, or release following addition of K+ at a concentration of 20 mM, or 35 mM. 6. Verapamil (25mgkg-', i.p.), nicardipine (25mgkg-1, i.p.) and nifedipine (20mgkg-1, i.p.) all markedly inhibited the 5-HT2 receptor-mediated head twitch response in mice produced by injection of 5- methoxy-N,N-dimethyl-tryptamine (5-MeODMT). Felodipine had the same effect with an ED50 of 2.6mgkg-'. Bay K 8644 did not reverse this effect. Both verapamil (IC50:2.5 microM) and nicardipine (IC50:8 microM) were 5-HT2 antagonists as indicated by inhibition of [3H]-ketanserin binding in mouse frontal cortex. However felodipine and nifedipine antagonized 5-HT2 receptor binding only in the millimolar range.7. Hydralazine (5mg kg 1, i.p.) induced the 5-HT behavioural syndrome in tranylcypromine pretreated rats, enhanced the tranylcypromine/L-tryptophan behavioural syndrome, inhibited 5-MeODMT-induced head twitch behaviour in mice and was not a 5-HT2 receptor antagonist. 8. These data indicate that at a high dose, Ca2+ antagonists produce complex changes in 5-HT function in rodents which are similar to those produced by lithium administration. The data with hydralazine suggest that the effects seen are not related to an action at Ca2 + channels.

Differential effects of tranylcypromine and imidazole on mammary carcinogenesis in rats fed low and high fat diets.

Neoplastic development in the rat mammary gland can be suppressed by inhibition of the activity of several enzymes involved in eicosanoid biosynthesis. In order to investigate the potential utility of prostacyclin and thromboxane synthetases as targets for mammary cancer chemoprevention, experiments were conducted to determine the influence of tranylcypromine (TCP), an inhibitor of prostacyclin synthetase, and imidazole (IMI), an inhibitor of thromboxane synthetase, on mammary carcinogenesis induced in rats by N-methyl-N-nitrosourea. Fifty-day-old female Sprague-Dawley [Hsd:SD(BR)] rats received a single s.c. dose of 0 or 40 mg of N-methyl-N-nitrosourea per kg of body weight. Beginning 7 days after carcinogen administration, groups of rats were fed isoenergetic, casein-based diets containing 3 or 20% corn oil (w/w), supplemented with (per kg of diet) 10 mg of TCP, 1000 mg of IMI, or sucrose carrier only. TCP reduced mammary carcinoma multiplicity in rats fed the 20% corn oil diet, but had no effect in rats fed the diet containing 3% fat. By contrast, supplementation with IMI increased mammary cancer incidence in the group fed the 20% fat diet and increased carcinoma multiplicity in the 3% fat group to the levels seen in rats fed the 20% fat diet. These data suggest that inhibition of prostacyclin synthetase, but not thromboxane synthetase, may present a useful mechanism for mammary cancer chemoprevention in animals consuming a diet high in fat. Furthermore, the differential effects of TCP and IMI in rats fed low and high fat diets suggest that the action of dietary fat in mammary cancer induction may involve influences on the arachidonic acid cascade.

Comparison of the effects of selected drugs on the release of hypothalamic adrenaline and noradrenaline measured in vivo.

Intracerebral dialysis combined with high-performance liquid chromatography and electrochemical detection was used to monitor changes in extracellular posterior hypothalamic, noradrenaline, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindole acetic acid (5-HIAA) following administration of an inhibitor of phenylethanolamine-N-methyltransferase (LY87130); the alpha 2-antagonist idazoxan; the monoamine oxidase (MAO) inhibitor tranylcypromine, and a selective noradrenergic neurotoxin (DSP4) to the anaesthetised rat. LY87130 (50 mg/kg i.p.) decreased basal hypothalamic perfusate and whole-tissue levels of adrenaline by 100% and 64%, respectively, but was without effect on basal extracellular hypothalamic levels and whole-tissue levels of noradrenaline, DOPAC and 5-HIAA. Administration of the alpha 2-adrenoceptor antagonist idazoxan and the MAO inhibitor tranylcypromine elicited increases in hypothalamic extracellular levels of both adrenaline and noradrenaline by 208% and 229%, respectively. Idazoxan also increased hypothalamic extracellular 5-HIAA by 97% but was without effect on extracellular DOPAC. In contrast, tranylcypromine decreased hypothalamic extracellular levels of DOPAC and 5-HIAA by 72% and 50%, respectively. DSP4 depleted extracellular hypothalamic adrenaline and noradrenaline 360 min and 390 min postdrug, respectively, after causing an initial 3-fold increase in both these amines 150 min after drug administration. DSP4 was without effect on posterior hypothalamic extracellular DOPAC and 5-HIAA. These results demonstrate that the pharmacology of central adrenaline and noradrenaline is very similar and, with the exception of phenylethanolamine-N-methyl transferase inhibitors, none of the drugs investigated are able to differentiate between adrenergic and noradrenergic neurones.

Adrenaline in the CNS: in vivo evidence for a functional pathway innervating the hypothalamus.

This article reviews the evidence, obtained from studies employing the technique of intracerebral dialysis, to monitor changes in extracellular adrenaline in the hypothalamus, that adrenaline is released from nerve endings in the hypothalamus and has a functional role. Studies using [3H] adrenaline to determine the specificity of uptake mechanisms for adrenaline indicate that labelled adrenaline is also taken up by noradrenergic nerve endings. This supports the need to develop techniques to monitor changes in endogenous release of adrenaline. In this study, it has been shown that inhibition of phenylethanolamine-N-methyltransferase selectively decreases tissue levels in the hypothalamus and in vivo release of adrenaline, while monoamine oxidase inhibition and antagonism of alpha 2-adrenoceptors increases the release of both adrenaline and noradrenaline. The "selective" noradrenergic neurotoxin DSP4 caused an initial increase in the release of both catecholamines, followed by a marked decrease in their release. Stimulation of the adrenaline-containing neurones in the rostral ventrolateral medulla (C1 region) increased the release of adrenaline in the posterior hypothalamus, but not that of noradrenaline, while also increasing mean arterial blood pressure. Pharmacological evidence indicates that B2-adrenoceptors in the hypothalamus and the spinal cord are involved in the rise in mean arterial pressure, as the response is reduced by a selective B2-antagonist (ICI 118551). The rise in mean arterial blood pressure during C1 stimulation is enhanced by the alpha 2-antagonist idazoxan, supporting observations that alpha 2-adrenoceptors are involved in the pre-synaptic regulation of release of adrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)