Loop diuretics decrease the renal elimination rate and increase the plasma levels of trimethylamine-N-oxide.

AIMS: Trimethylamine-N-oxide (TMAO) is a novel cardiovascular risk marker. We explored the association of commonly used cardiovascular medications with TMAO levels in patients and validated the identified associations in mice. METHODS: Detailed history of drug treatment was recorded in 300 patients with cardiovascular disease without diabetes in an observational, cross-sectional study. Animal study was performed in CD1 mice. RESULTS: Median plasma TMAO (interquartile range) level was 2.144 (1.570-3.104) µmol l-1 . Among nine cardiovascular drug groups, the use of loop diuretics (0.510 ± 0.296 in users vs. 0.336 ± 0.272 in nonusers, P = 0.008) and mineralocorticoid receptor antagonists (0.482 ± 0.293 in users vs. 0.334 ± 0.272 in nonusers, P = 0.007) was associated with increased log-TMAO. Acute concomitant administration of furosemide or torasemide with TMAO in mice significantly influenced TMAO pharmacokinetic profile and almost doubled the plasma TMAO area under the curve. Furosemide decreased the TMAO excretion rate by 1.9-fold during the first 30 min after administration and increased TMAO concentrations in kidney, heart and liver, suggesting the interaction of furosemide and TMAO with efflux transporters. The concentrations of TMAO in blood plasma after the administration of the organic anion transporter inhibitor probenecid were not different from those of the control group, suggesting an effect not mediated by organic anion transporters. CONCLUSIONS: Loop diuretics increased plasma TMAO concentration by decreasing its urinary excretion rate. Loop diuretic use should be considered a potential confounder in TMAO studies.

The Differentiation of Skin Mesenchymal Stem Cells Towards a Schwann Cell Phenotype: Impact of Sigma-1 Receptor Activation.

Neural crest stem cells (NCSCs) are the source of mature Schwann cells in the peripheral nervous system (PNS). The NCSC population resides in the bulge of hair follicles and in the dermis. Recently, it was shown that 2-3% of the human dermis mesenchymal stem cell (MSC) population expresses the NCSC marker CD271, thus enabling the use of skin MSCs for studying Schwann cell differentiation in vitro. The aims of this study were to establish a protocol for human skin MSC differentiation towards Schwann cell-like cells (SC-lcs) and to analyse the expression of sigma-1 receptor (S1R) in SC-lcs. The impact of S1R ligands, namely the selective agonist PRE-084, the positive allosteric modulator E1R and the selective antagonist NE-100, on Schwann cell differentiation was assessed. The expression of the neuron-specific genes Tubulin-ßIII and Integrin-6α, the Schwann cell-specific gene S100b, MBP and the NCSC-specific genes p75NTR, Sox10, Notch1, Integrin-4α, Ap2α and Pax6 was analysed in MSCs and SC-lcs by real-time RT-PCR. BDNF secretion was evaluated by ELISA. The effect of S1R ligands on SC-lc differentiation was measured using BDNF ELISA and MBP flow cytometry. After MSC differentiation, NCSC markers p75NTR and Integrin-4α were downregulated 3.5-fold and 2-fold, respectively. To the contrary, MBP and S100b were significantly upregulated in SC-lcs. S1R ligands showed a tendency to increase the secretion of BDNF by the SC-lc population. PRE-084 and E1R increased MBP expression in the SC-lc population, whereas 3 µM NE-100 inhibited MBP expression in SC-lcs. In conclusion, our data demonstrate that S1R plays an important role in skin MSC differentiation towards myelinating Schwann cells.

Diabetes is Associated with Higher Trimethylamine N-oxide Plasma Levels.

Recent studies have revealed strong associations between systemic trimethylamine N-oxide (TMAO) levels, atherosclerosis and cardiovascular risk. In addition, plasma L-carnitine levels in patients with high TMAO concentrations predicted an increased risk for cardiovascular disease and incident major adverse cardiac events. The aim of the present study was to investigate the relation between TMAO and L-carnitine plasma levels and diabetes. Blood plasma samples were collected from 12 and 20 weeks old db/db mice and patients undergoing percutaneous coronary intervention. Diabetic compared to non-diabetic db/L mice presented 10-fold higher TMAO, but lower L-carnitine plasma concentrations at 12 weeks of age. After 8 weeks of observation, diabetic db/db mice had significantly increased body weight, insulin resistance and TMAO concentration in comparison to non-diabetic control. In 191 patients undergoing percutaneous coronary intervention the median (interquartile range) plasma concentration of TMAO was 1.8 (1.2-2.6) µmol/L. Analysis of the samples showed a bivariate association of TMAO level with age, total cholesterol and L-carnitine. The multivariate linear regression analysis revealed that, in addition to L-carnitine as the strongest predictor of log transformed TMAO (p<0.001), the parameters of age, diabetes status and body mass index (BMI) were independently associated with increased log transformed TMAO levels (p<0.01).Our data provide evidence that age, diabetes and BMI are associated with higher TMAO levels independently of L-carnitine. These data support the hypothesis of TMAO as a cardiovascular risk marker and warrant further investigation of TMAO for diabetes research applications.

Inhibition of L-carnitine biosynthesis and transport by methyl-γ-butyrobetaine decreases fatty acid oxidation and protects against myocardial infarction.

BACKGROUND AND PURPOSE: The important pathological consequences of ischaemic heart disease arise from the detrimental effects of the accumulation of long-chain acylcarnitines in the case of acute ischaemia-reperfusion. The aim of this study is to test whether decreasing the L-carnitine content represents an effective strategy to decrease accumulation of long-chain acylcarnitines and to reduce fatty acid oxidation in order to protect the heart against acute ischaemia-reperfusion injury. KEY RESULTS: In this study, we used a novel compound, 4-[ethyl(dimethyl)ammonio]butanoate (Methyl-GBB), which inhibits γ-butyrobetaine dioxygenase (IC50 3 µM) and organic cation transporter 2 (OCTN2, IC50 3 µM), and, in turn, decreases levels of L-carnitine and acylcarnitines in heart tissue. Methyl-GBB reduced both mitochondrial and peroxisomal palmitate oxidation rates by 44 and 53% respectively. In isolated hearts treated with Methyl-GBB, uptake and oxidation rates of labelled palmitate were decreased by 40%, while glucose oxidation was increased twofold. Methyl-GBB (5 or 20 mg·kg(-1)) decreased the infarct size by 45-48%. In vivo pretreatment with Methyl-GBB (20 mg·kg(-1)) attenuated the infarct size by 45% and improved 24 h survival of rats by 20-30%. CONCLUSIONS AND IMPLICATIONS: Reduction of L-carnitine and long-chain acylcarnitine content by the inhibition of OCTN2 represents an effective strategy to protect the heart against ischaemia-reperfusion-induced damage. Methyl-GBB treatment exerted cardioprotective effects and increased survival by limiting long-chain fatty acid oxidation and facilitating glucose metabolism.

Risks and benefits of carnitine supplementation in diabetes.

L-carnitine is a very popular food supplement due to its safety profile, antioxidant-type activity and suggested effects on energy metabolism pathways. L-carnitine participates in both fatty acid transport pathways and the export of acetyl groups out of the mitochondria. However, contradictory data exist concerning the pharmacological outcomes of L-carnitine treatment in diabetes mellitus, which is a highly prevalent metabolic disease characterised by hyperglycemia and associated with severe complications, including cardiovascular disease and dyslipidemia. Recently, the L-carnitine-derived metabolites, acylcarnitines and trimethylamine-N-oxide, have been associated with increased cardio-metabolic risks. This review aims to highlight the possible risks and benefits of L-carnitine supplementation.

[Carnitine as a marker of atherosclerosis and other risks of cardiovascular diseases].

L-carnitine was first isolated from the extracts of muscle tissue in 1905 by the employees of the department of medicinal chemistry at Moscow University. Later the role of L-carnitine in both the oxidation of long chain fatty acids and the metabolism of carbohydrates was discovered. Today L-carnitine is not just a drug for the treatment of pathologies associated with its deficiency, but also a widespread dietary supplement, believed to be able to reduce weight and improve the physical qualities of a person. However, in light of the recent findings about a possible link between L-carnitine and the development of atherosclerosis, a careful assessment of the use of L-carnitine as a safe dietary supplement is required, particularly when there is no sound medical indication.

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.

In vivo measurements of the cerebral perfusion and cardiovascular effects of the novel guanidine ME10092 in the non-human primate, Papio ursinus.

The novel guanidines N-(3,4-dimethoxy-2-chlorobenzylideneamino)-guanidine (ME 10092) and N-(3,4-dimethoxy-2-chlorobenzylideneamino)-N1-hydroxyguanidine (PR5) were recently reported to exhibit promising cardioprotective activities in myocardial ischaemia and reperfusion in rats. The current study investigated for the first time pharmacological effects of ME10092 in the primate, viz. the Cape baboon Papio ursinus. The effects of ME10092 (1 and 2 mg/kg doses) on the cerebral blood flow, heart rates and the systolic and diastolic blood pressure were investigated after intravenous injection to the baboon under anaesthesia. The cerebral perfusion effects of ME10092 were assessed using Single Photon Emission Computed Tomography according to the split-dose approach and 99mTc-hexamethyl-propylene amine oxime as brain perfusion tracer. The observation that the recovery times from the anaesthesia were unacceptably prolonged excluded doses beyond 2 mg/kg. The data indicate that no cerebral perfusion changes were induced at both the 1 and 2 mg/kg doses of ME10092. Both these doses of ME10092 showed blood pressure and heart rate effects, with the latter being more significant. Decreases in heart rate were seen directly after ME10092 administration reaching levels of about 20% for the 2 mg/kg dose and about 15% for the 1 mg/kg dose at around 6 min post drug administration. A transient decrease in both systolic and diastolic blood pressure was observed for the higher dose. The blood pressure data further suggest an attenuation of the anaesthesia induced increase in pressure usually present in non-intervention studies. ME10092 clearly exhibits mycocardial effects in the non-human primate, similar to the effects previously observed in the ischaemia-reperfusion rat model, where ME10092 showed strong protection.

[Biochemical mechanisms of mildronate action during ischemic stress]. / Biokhimicheskie mekhanizmy deistviia mildronata v usloviiakh ishemicheskogo stressa.

The authors in the article described biochemical mechanisms of Mildronat. The main mechanism of Mildronat activity is based on decreasing carnitine level in the organism, which leads to hampering oxidation of fatty acids. Mildronat is believed keeps on training myocardium pharmacologically even without physical activity by adapting cells to decreasing fat acids inflow and activating glucose oxidations. Under ischemic condition to obtain energy, cells use intensively glucose oxidation. Clinical studies have reliably showed Mildronat to have positive effect in treating patients with cardiovascular and ischemic cerebral diseases as well as for enhancing physical and mental efficiency.

EPR investigation of in vivo inhibitory effect of guanidine compounds on nitric oxide production in rat tissues.

The aim of the present study was to evaluate in vivo effects on NO production of pharmacologically widely used, commercially available NOS inhibitors, structurally related to guanidine. We compared the NO inhibitory potency and selectivity of L-NAME, aminoguanidine and guanabenz in tissues of normal and LPS-stimulated rats using ex vivo EPR measurements of the NO radical in its complex with dithiocarbamate-Fe(II). The tissues studied were the brain cortex, kidney, liver, heart and testis. Differential inhibitory effects were seen for L-NAME, aminoguanidine and guanabenz when applied during basal or LPS-stimulated conditions. Aminoguanidine exerted inhibition of NO only after stimulation with LPS. Guanabenz had little effect on NO in liver, kidney, testis and heart under normal conditions, while it reduced the basal NO in brain cortex. After stimulation with LPS guanabenz afforded a partial inhibition of the NO formation in all tissues studied. L-NAME was a potent inhibitor of NO synthesis in all tested tissues, both during basal and LPS stimulated conditions. Our results show that compounds containing a guanidine moiety might possess different NOS inhibitory profiles in vivo.

Improved method for EPR detection of DEPMPO-superoxide radicals by liquid nitrogen freezing.

5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) is frequently used as a spin trap for the measurement of superoxide by EPR spectrometry. However, its half life is fairly short in room temperature. We here show that superoxide radicals trapped by DEPMPO can be successfully recorded at -196 degrees C. Moreover, we show that the signal intensity remains unaltered for up to 7 days, when the samples are stored in liquid nitrogen. Our new approach for measurement of superoxide should greatly simplify the studies of this important radical in biological systems.

N-Hydroxyguanidine compound 1-(3,4-dimethoxy- 2-chlorobenzylideneamino)-3-hydroxyguanidine inhibits the xanthine oxidase mediated generation of superoxide radical.

We here show that the novel N-hydroxyguanidine derivative PR5 (1-(3, 4-dimethoxy-2-chlorobenzylideneamino)-3-hydroxyguanidine) is acting as an alternative electron acceptor in xanthine oxidase catalyzed oxidation of xanthine. The reduction product is the corresponding guanidine derivative 1-(3, 4-dimethoxy-2-chlorobenzylideneamino)guanidine (PR9). The reaction occurs under both anaerobic and aerobic conditions. Moreover, EPR measurements show that the action of PR5 is associated with the inhibition of superoxide radical formation seen under aerobic conditions. PR5 also supports xanthine oxidase catalyzed anaerobic oxidation of NADH. Kinetic studies indicate that increasing xanthine concentration significantly increases the apparent K(m) of PR5, but it remains unaltered by changing NADH concentration. Moreover, the molybdenum center inhibitor allopurinol inhibits the PR5-sustained oxidation of xanthine and NADH equally well, whereas the flavin adenine dinucleotide site inhibitor diphenyliodonium (DPI) markedly inhibits only the PR5-sustained oxidation of NADH. We suggest that PR5 binds and becomes reduced at the molybdenum center of the xanthine oxidase. We also found that both PR5 and its reduction product PR9 can inhibit the oxygen-sustained xanthine oxidase reaction. The properties of PR5 suggest that it is a member of a novel class of compounds which we have termed xanthine oxidase electron acceptor-inhibitor drugs. The potential use of xanthine oxidase electron acceptor-inhibitors in the prevention of free radical mediated tissue damage in organ ischemia-reperfusion diseases is discussed.

Cardioprotective effects of N-hydroxyguanidine PR5 in myocardial ischaemia and reperfusion in rats.

1. The potential for the N-hydroxyguanidine compound PR5 (N-(3, 4-dimethoxy-2-chlorobenzylideneamino)-N'-hydroxyguanidine) as a cardioprotective agent in heart ischaemia and reperfusion injury was investigated using rat models. 2. Administration of 1-10 mg kg-1 of PR5 5 min before 10 min of left coronary artery occlusion, followed by 20 min reperfusion, strongly inhibited reperfusion burst of arrhythmias and markedly improved the survival of the animals (e.g. ventricular fibrillation incidence 93 vs 43% (P<0.05); mortality 47 vs 0% (P<0.05), for controls and for 3 mg kg-1 of PR5, respectively). 3. Administration of 3 mg kg-1 of PR5 1 min before reperfusion to rats subjected to 10 min occlusion, 20 min reperfusion was most effective in reducing arrhythmias and decreasing mortality (43 vs 0%, P<0.05), but effects were also seen when PR5 was administered 0, 1 and 5 min after start of reperfusion. 4. Coronary occlusion/reperfusion (10 - 20 min) increased malondialdehyde (MDA) of rat hearts (0.88+/-0.13 for sham vs 1.45+/-0.12 nmol mg-1 protein for ischaemic; P<0.05). In rats where 3 mg kg-1 PR5 were administered 1 min before reperfusion the increase was attenuated (MDA being 1.04+/-0.12; P<0.05 vs ischaemic). 5. PR5 caused a substantial reduction of the infarction size in rats subjected to 180 min left coronary artery occlusion, followed by 120 min of reperfusion; the necrotic zone being 326+/-32 mg for controls vs 137+/-21 mg for animals treated with 3x3 mg kg-1 of PR5 (P<0.01). 6. PR5 reduced the elevation of the ST-segment of the ECGs, as well as caused pronounced attenuation of the rapid blood pressure drop seen at the start of reperfusion following coronary artery occlusion. 7 We conclude that the N-hydroxyguanidine PR5 provides remarkable protection against ischaemia and reperfusion induced myocardial necrosis and life-threatening arrhythmias. These effects of PR5 are discussed in relation to a recently discovered ability of N-hydroxyguanidines to function as electron acceptors at the xanthine oxidase enzyme.

Identification of an N-hydroxyguanidine reducing activity of xanthine oxidase.

A guanoxabenz [1-(2,6-dichlorobenzylideneamino)-3-hydroxyguanidine; an N-hydroxyguanidine] reducing enzymatic activity of rat spleen cytosol was investigated. By means of protein purification and N-terminal amino acid sequencing, the reducing activity was shown to reside in xanthine oxidase. The action of the enzyme on guanoxabenz resulted in the formation of guanabenz [1-(2,6-dichlorobenzylidene-amino)-3-guanidine]; the product formation could be monitored by HPLC and its identity was confirmed by NMR analysis. The reduction of guanoxabenz required xanthine or NADH as reducing substrates, while the process could be blocked by allopurinol, a selective inhibitor of xanthine oxidase. By using bovine milk xanthine oxidase, the guanoxabenz reducing activity of the enzyme was also verified. We conclude that guanoxabenz is a novel electron acceptor structure for xanthine oxidase.

Characterization of guanoxabenz reducing activity in rat brain.

Guanoxabenz (1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine) and guanabenz (1-(2,6-dichlorobenzylidene-amino)-3-guanidine) are both known as centrally active antihypertensive drugs. We have previously shown that enzymatic activity in the rat spleen can induce N-reduction of guanoxabenz, leading to high affinity alpha 2-adrenoceptor binding, due to the formation of the alpha 2-adrenoceptor active drug, guanabenz. The spleen activity appears to reside in xanthine oxidase as it is activated by xanthine and blocked by allopurinol. We report that high affinity guanoxabenz binding is also induced in rat brain membranes after addition of NADH or NADPH cofactors. However, the brain process was clearly different from that of the spleen, as the formation of high affinity binding in the brain was not blocked by allopurinol. Moreover the NADH/NADPH activated mechanism of the brain membranes was not blocked by carbon monoxide and SKF525A, thus the activity appears not to reside in cytochrome P450 enzymes. Instead the activity was blocked by menadione and dicumarol. We conclude that the rat cerebral cortex contains an enzymatic activity that may activate guanoxabenz leading to formation of a metabolite showing high affinity for alpha 2-adrenoceptors. We also conclude that the rat brain activity is clearly distinct from that of the rat spleen.

Characterization of the enzymatic activity for biphasic competition by guanoxabenz (1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine) at alpha2-adrenoceptors. II. Description of a xanthine-dependent enzymatic activity in spleen cytosol.

The mechanism for formation of high affinity binding of guanoxabenz (1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine) to alpha2-adrenoceptors by the rat spleen cytosol was studied. We report here that the spleen cytosolic fraction mediated the reduction of guanoxabenz to guanabenz (1-(2,6-dichlorobenzylidene-amino)-3-guanidine), the latter having an almost 100-fold higher affinity for rat alpha2A-adrenoceptors than guanoxabenz itself. The reaction product could be separated by high-performance liquid chromatography and its identity as guanabenz confirmed by nuclear magnetic resonance. The spleen cytosolic activity could be separated into high and low molecular weight components, the high molecular weight component requiring low molecular weight factors for maximal activity. Xanthine oxidase seems to be the most likely candidate responsible for the activity, as the guanoxabenz-reducing activity of the high molecular weight component could be sustained by exogenously applied xanthine, while it was potently blocked by allopurinol. The conversion of guanoxabenz by the cytosolic activity was also quite potently blocked by DWO1, 1-(3,4-dimethoxybenzylideneamino)3-hydroxyguanidine, a hydroxyguanidine analogue to guanoxabenz.

Characterization of the enzymatic activity for biphasic competition by guanoxabenz (1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine) at alpha2-adrenoceptors. I. Description of an enzymatic activity in spleen membranes.

The mechanism for formation of high-affinity binding of 1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine (guanoxabenz) to alpha2-adrenoceptors was studied in particulate fractions from the rat spleen. The proportion of apparent high versus low-affinity alpha2-adrenoceptor binding sites increased with increasing incubation time and was also augmented by Mg2+ ions. The formation of high-affinity guanoxabenz binding seemed to be inhibited by a series of N-hydroxyguanidine analogs to guanoxabenz, as well as by a series of metabolic inhibitors that included allopurinol, 1-chloro-2,4-dinitrobenzene, 5,5'-dithiobis-(2-nitrobenzoic acid), cibacron blue, phenyl-p-benzoquinone, didox, and trimidox. The formation of guanoxabenz high-affinity binding was also inhibited in a time- and concentration-dependent fashion by preincubating the membranes with the LW03 N-hydroxyguanidine analogue of guanoxabenz. Moreover, when the spleen membranes were extensively washed for 30 min with buffers at 25 degrees, the guanoxabenz high-affinity binding disappeared. However, when these washed membranes were supplemented with xanthine, the apparent affinity of guanoxabenz increased four to five-fold. Taken together, all data were compatible with the theory that the formation of high-affinity binding was dependent on the generation of a guanoxabenz metabolite that showed an approximate 100-fold greater affinity for the alpha2-adrenoceptors than guanoxabenz itself. Because the most potent blocker of the formation of high-affinity binding was allopurinol (apart from some N-hydroxyguanidine analogs to guanoxabenz) and since the activity could be restored with xanthine, a likely candidate responsible for the metabolic activation is xanthine oxidase.

Conditions for biphasic competition curves in radioligand binding for ligands subjected to metabolic transformation.

The conditions affording biphasic competition curves in radioligand binding for ligands subjected to metabolic transformation was analyzed theoretically. It was shown that when a competing ligand was subjected to transformation to a ligand that showed higher affinity than the parent compound, biphasic competition curves, which might wrongly be interpreted as indicating the presence of two receptor sites, could be observed in binding assays containing a homogenous receptor population. Biphasic competition curves were seen if the conversion of the competitor occurred according to zero and second order kinetics, as well as by enzymatic catalytic processes. However, when the conversion occurred according to a first order kinetics, the competition curves were uniphasic and resolved only into one-site fits, with the apparent affinity of the competitor reflecting the degree of conversion of the competitor to its metabolite. When the metabolic conversion resulted in a metabolite that showed lower affinity for the receptor than the parent compound, the competition curves became supersteep for conversions according to zero and second order kinetics, as well as for conversion by enzymatic catalytic processes.

[3H]RS79948-197 binding to human, rat, guinea pig and pig alpha2A-, alpha2B- and alpha2C-adrenoceptors. Comparison with MK912, RX821002, rauwolscine and yohimbine.

The Kd values of the recently introduced radioligand [3H]RS79948-197 ((8a R,12aS,13a-S)-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-metho xy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]naphthyridine) were determined for the recombinant human and rat alpha2A-, alpha2B- and alpha2C- as well as guinea pig alpha2B- and alpha2c-adrenoceptors expressed in COS (CV-1 Origin, SV40) cells. In addition, the Kd values were also determined for [3H]RS79948-197 for the guinea pig spleen alpha2A-adrenoceptor and for pig alpha2A-, alpha2B- and alpha2C-adrenoceptors in membranes obtained from kidney and striatum. Available radioligands for alpha2-adrenoceptors, besides [3H]RS79948-197 are the tritiated forms of MK912 ((2S,12bS)1',3'-dimethylspiro(1,3,4,5',6,6',7,12b-octa hydro-2H-benzo[b]furo[2,3-a]quinazoline)-2,4'-pyrimidin-2'-one), RX821002 (2-methoxy-idazoxan), rauwolscine and yohimbine. In the present article the binding constants of all these substances for the alpha2A-, alpha2B- and alpha2C-adrenoceptor subtypes in human, pig, rat and guinea pig are reviewed. In all species tested MK912 was alpha2C-selective, RX821002 showed a minor alpha2A-selectivity, whereas [3H]RS79948-197 was non-selective among the alpha2-adrenoceptor subtypes, showing high affinity for all three subtypes. Rauwolscine and yohimbine showed relatively low affinities for nmost of the alpha2-adrenoceptor subtypes investigated, the exception being rauwolscine having high affinity for the human and porcine alpha2C-adrenoceptors.

"Atypical" neuromodulatory profile of glutapyrone, a representative of a novel 'class' of amino acid-containing dipeptide-mimicking 1,4-dihydropyridine (DHP) compounds: in vitro and in vivo studies.

Glutapyrone, a disodium salt of 2-(2,6-dimethyl-3,5-diethoxycarbonyl-1,4-dihydropyridine-4-carboxamido)- glutaric acid, is a representative of a novel 'class' of amino acid-containing 1,4-dihydropyridine (DHP) compounds developed at the Latvian Institute of Organic Synthesis, Riga, Latvia. Conceptually, the glutapyrone molecule can be regarded as a dipeptide-mimicking structure formed by the "free" amino acid (glutamate) moiety and "crypto" (built into the DHP cycle) amino acid ("GABA") elements. Both of these amino acids are joined by the peptide bond. This compound unlike classical DHPs lacks calcium antagonistic or agonistic properties. Our previous studies revealed a profound and long-term anticonvulsant, stress-protective and neurodeficit-preventive activities of glutapyrone. In view of structural properties the role of glutamatergic mechanisms in the mediation of central effects of glutapyrone was considered. In the present study glutapyrone at the concentration range of 1 microM(-1) mM failed to effect both NMDA ([3H]TCP) and non-NMDA ([3H]KA and [3H]AMPA) receptor ligand binding in the rat cortical membranes in vitro. The compound markedly enhanced motor hyperactivity induced by the NMDA antagonist PCP and the dopamine releasing compound D-amphetamine in the rats. Glutapyrone displayed activity in a variety of animal models relevant for affective/depressive disorders in humans i.e. reserpine-induced ptosis and hypothermia, forced swimming test and open field test. These data indicate that the unusually "broad" pharmacological spectrum of glutapyrone might involve concomitant actions on multiple neurotransmitter systems, particularly, GABA-ergic and the catecholamines. It is discussed whether these functional properties are secondary to action on intracellular events, predominantly, G protein-related since glutapyrone appears to lack direct interactions with a number of receptors including ionotropic glutamate and GABA(A)/Bzd receptors.