Carbamylation reduces the capacity of IgG for hexamerization and complement activation.

Carbamylation is a post-translational modification that can be detected on a range of proteins, including immunoglobulin (Ig)G, in several clinical conditions. Carbamylated IgG (ca-IgG) was reported to lose its capacity to trigger complement activation, but the mechanism remains unclear. Because C1q binds with high affinity to hexameric IgG, we analyzed whether carbamylation of IgG affects binding of C1q, hexamerization and complement-dependent cytotoxicity (CDC). Synovial tissues of rheumatoid arthritis (RA) patients were analyzed for the presence of ca-IgG in vivo. Synovial tissues from RA patients were analyzed for the presence of ca-IgG using mass spectrometry (MS). Monomeric or hexameric antibodies were carbamylated in vitro and quality in solution was controlled. The capacity of ca-IgG to activate complement was analyzed in enzyme-linked immunosorbent (ELISAs) and cellular CDC assays. Using MS, we identified ca-IgG to be present in the joints of RA patients. Using in vitro carbamylated antibodies, we observed that ca-IgG lost its capacity to activate complement in both solid-phase and CDC assays. Mixing ca-IgG with non-modified IgG did not result in effective inhibition of complement activation by ca-IgG. Carbamylation of both monomeric IgG and preformed hexameric IgG greatly impaired the capacity to trigger complement activation. Furthermore, upon carbamylation, the preformed hexameric IgG dissociated into monomeric IgG in solution, indicating that carbamylation influences both hexamerization and C1q binding. In conclusion, ca-IgG can be detected in vivo and has a strongly reduced capacity to activate complement which is, in part, mediated through a reduced ability to form hexamers.

[Basophilic granulocytes and autoimmune diseases. Can basophilic granulocytes modulate B-cell functions in systemic lupus erythematosus?]. / Basophile Granulozyten und Autoimmunerkrankungen. Können basophile Granulozyten die Funktion von B-Zellen beim systemischen Lupus erythematodes beeinflussen?

BACKGROUND: B-cells play an important role in the pathogenesis of systemic lupus erythematosus (SLE); however, many other cell types are also involved in disease development. In a murine lupus model it was demonstrated that basophils are indispensable for the development of lupus symptoms. AIM: This study investigated whether there is evidence for a relevant interaction between B-cells and basophils under physiological and pathological conditions. MATERIAL AND METHODS: A selective review of the literature was performed and some preliminary data about the interaction of basophils and B-cells are reported in this article. For the experiments, isolated B-cells were cultured in vitro in the presence or absence of basophils and B-cell survival, proliferation, plasma cell development and antibody production were determined. RESULTS: Data from the literature show that there is evidence for an interaction between basophils and B-cells in a murine model. Our investigations confirmed that human basophils also support the survival and proliferation of B-cells. Furthermore, plasma cell differentiation and antibody production, most importantly IgG secretion, are enhanced. First experimental ex vivo analyses of basophils from SLE patients demonstrate that these cells exhibit a higher activation level compared to basophils from healthy controls. DISCUSSION: In summary, previously published data and our own data demonstrate that there is an interaction between human basophils and B-cells. A better understanding of the role of basophils in the pathogenesis of SLE could lead to the development of new therapeutic strategies.

Murine and human Langerhans cells express a functional histamine H4 receptor: modulation of cell migration and function.

BACKGROUND: Histamine is an important mediator of allergic reactions, and recent studies indicated that the function of different types of antigen presenting cells (APC) can be modulated by histamine, in particular via the newly described histamine H(4) receptor (H(4)R). Therefore, we investigated possible interactions of histamine via the H(4)R on Langerhans cells (LC), which represent the professional APC in the skin and therefore have an important role in the initiation and maintenance of allergic skin diseases. METHODS: The expression of the H(4)R was evaluated by real-time PCR, flow cytometry and immunofluorescence staining. The function of the H(4)R was determined by intracellular flow cytometric measurement of chemokine production and LC migration assays. RESULTS: Here, we show H(4)R expression on in vitro generated monocyte-derived LC (mRNA and protein) and on primary LC from murine and human skin samples (protein). The immunofluorescence staining in murine and human skin samples clearly proved that LC express the H(4)R in situ. Stimulation with histamine or a H(4)R agonist downregulated the chemokine (C-C motif) ligand 2 (CCL2) in human monocyte-derived LC and primary LC. Prestimulation with a selective H(4)R antagonist abolished this effect. Moreover, migration of LC from the epidermis was increased after H(4)R agonist stimulation in ex vivo migration assays using human epidermis and murine in vivo assays. CONCLUSION: Our findings show that LC express a functional H(4)R and point towards a possible pathogenic relevance of the H(4)R in inflammatory and allergic diseases.

Tough combinatorial poly(urethane-isocyanurate) polymer networks and hydrogels synthesized by the trimerization of mixtures of NCO-prepolymers.

The development of tough hydrogels is an essential but challenging topic in biomaterials research that has received much attention over the past years. By the combinatorial synthesis of polymer networks and hydrogels based on prepolymers with different properties, new materials with widely varying characteristics and unexpected properties may be identified. In this paper, we report on the properties of combinatorial poly(urethane-isocyanurate) (PUI) type polymer networks that were synthesized by the trimerization of mixtures of NCO-functionalized poly(ethylene glycol) (PEG), poly(propylene gylcol) (PPG), poly(ε-caprolactone) (PCL) and poly(trimethylene carbonate) (PTMC) prepolymers in solution. The resulting polymer networks showed widely varying material properties. Combinatorial PUI networks containing at least one hydrophilic PEG component showed high water uptakes of >100 wt%. The resulting hydrogels demonstrated elastic moduli of up to 10.1 MPa, ultimate tensile strengths of up to 9.8 MPa, elongation at break values of up to 624.0% and toughness values of up to 53.4 MJ m-3. These values are exceptionally high and show that combinatorial PUI hydrogels are among the toughest hydrogels reported in the literature. Also, the simple two-step synthesis and wide range of suitable starting materials make this synthesis method more versatile and widely applicable than the existing methods for synthesizing tough hydrogels. An important finding of this work is that the presence of a hydrophobic network component significantly enhances the toughness and tensile strength of the combinatorial PUI hydrogels in the hydrated state. This enhancement is the largest when the hydrophobic network component is crystallizable in nature. In fact, the PUI hydrogels containing a crystallizable hydrophobic network component are shown to be semi-crystalline in the water-swollen state. Due to their high toughness values in the water-swollen state together with their water uptake values, elastic moduli and ultimate tensile strengths, the developed hydrogels are expected to be promising materials for biomedical coating- and adhesive applications, as well as for tissue-engineering. STATEMENT OF SIGNIFICANCE: The development of tough hydrogels is a challenging topic that has received much attention over the past years. At present, double network type hydrogels are considered state-of-the-art in the field, demonstrating toughness values of several tens of MJ m-3. However, in terms of ease and versatility of the synthesis method, the possibilities are limited using a double network approach. In this work, we present combinatorial poly(urethane-isocyanurate) type polymer networks and hydrogels, synthesized by the trimerization of mixtures of NCO-functionalized prepolymers. The resulting hydrogels demonstrate exceptionally high toughness values of up to 53 MJ m-3, while the synthesis method is versatile and widely applicable. This new class of hydrogels is therefore considered highly promising in the future development of load-bearing biomaterials.

Histamine H4 receptors modulate dendritic cell migration through skin--immunomodulatory role of histamine.

BACKGROUND: Dendritic cells (DC) are the major antigen-presenting cells and play a key role in adaptive immunity as they are able to activate naive T cells. It was recently described, that the histamine H(4) receptor (H4R) is present on human monocyte-derived DC and that chemotaxis and T-helper (Th)1-Th2 polarization is mediated by this receptor. However, the distribution of histamine receptors on murine DC has not been studied yet. METHODS: The histamine receptor expression on murine bone marrow (BM)-derived DC and effects of histamine and H4R agonism on DC migration through skin were studied. As it was demonstrated in scratching experiments that NMRI mice are more susceptible to H4R-mediated itch than BALB/c mice, DC function of NMRI and BALB/c mice was compared. RESULTS: The mRNA of the H1R, H2R and H4R could be detected in murine BM-derived DC, while mRNA of the H3R was found to be low or undetectable. There were no distinct differences in mRNA expression and in H4R protein level (flow cytometry) between NMRI compared with BALB/c mice indicating, that a higher susceptibility is not associated with a generally higher H4R expression in all cell types. Histamine as well as the H4R agonist clobenpropit induced an enhanced chemotaxis in the skin DC migration assay. The enhanced chemotaxis was blocked by the H4R antagonist JNJ7777120. This finding was confirmed by in vitro migration experiments with BM-derived DC. CONCLUSION: Referring to DC migration, blocking the H4R on inflammatory cells might be a promising anti-inflammatory, immunomodulatory strategy.

Brain levels and metabolism of the dopaminergic agonist 2-amino-6,7-dihydroxytetrahydronaphthalene after administration of various prodrugs.

The synthesis of four prodrug diesters (diacetyl, diisobutyryl, dipivaloyl, and dibenzoyl) of the potent dopaminergic agonist 2-amino-6,7-dihydroxytetrahydronaphthalene (6,7-ADTN) is described. The effects of prodrug structure on the levels of 6,7-ADTN in the rat corpus striatum and cerebellum, as well as the levels of the metabolite, 6-hydroxy-7-methoxy-2-aminotetralin, in the corpus striatum, have been determined after intraperitoneal administration. In addition, the striatal levels of 6,7-ADTN after administration of the dibenzoyl analogue via intraperitoneal, subcutaneous, and oral routes have been measured. These prodrugs produce a significant improvement in the penetration and accumulation of 6,7-ADTN in the brain.

Synthesis of isomeric thienobenzothiazines and their effect on dopamine metabolism in rat brain.

The synthesis of the thieno[2,3-b]-, thieno[3,4-b]- and thieno[3,2-b]benzothiazines with a hydroxyethylpiperazinylpropyl side chain and various 2 substituents is described. The influence of these neuroleptic compounds on dopamine metabolism in vivo is quantitated by determining the rise in homovanillic acid concentrations in rat corpus striatum. Notable differences in activity were found among the isomers, which were useful for structure--activity correlations in the phenothiazine neuroleptics.

Facile syntheses of potent dopaminergic argonists and their effect on neurotransmitter release.

The facile syntheses of important intermediates used in the preparation of the two potent dopaminergic argonists, 2-amino-6,7-dihydroxytetrahydronaphthalene (11) (referred to by some authors as ADTN) and its 5,6-dihydroxyl isomer 12, are described. Thus 6,7-dimethyoxy-2-tetralone has been prepared in two steps and 5,6-dimethoxy-2-tetralone in three steps both from commercially available materials. The effects of 11, 12, and the noncatechol analogue, 2-aminotetrahydronaphthalene (ATN), on radioactive neurotransmitter release have been studied in vitro using rat brain slices. It has been shown that both 11 and 12, at a concentraiton of 2 micron, cause a release of [3H]-DA and NA, 11 being more potent than 12 in releasing [3H]-DA. ATN (2 micron) was found to be inactive in these experiments which shows the importance of the catechol function in this uptake--release process.

Synthesis and pharmacological evaluation of conformationally restricted phenothiazine analogues.

The synthesis of 3-(dimethylamino)-2,3-dihydro-4-chloro-1H-pyrido[3,2,1-kl]phenothiazine, its 10-chloro analogue, and two chloro isomers of 2[(dimethylamino)methyl]-2,3-dihydro-1H-pyrido[3,2,1-kl]phenothiazine is described. In these compounds the side chain of chlorpromazine is fixed into a certain position in order to study the dopamine-overlap theory and the role of the substituents in the phenothiazine neuroleptics. The compounds were tested for their influence on dopamine metabolism, while for the second set their ability to displace [3H]spiroperidol from dopamine receptors was assessed. No neuroleptic activity was found from the pharmacological data. The results are discussed on the basis of conformational requirements for dopamine antagonistic activity.

Synthesis and pharmacology of trans-4-n-propyl-3,4,4a,10b-tetrahydro-2H,5H-1-benzopyrano[4,3-b ]-1,4-oxazin-7- and -9-ols: the significance of nitrogen pKa values for central dopamine receptor activation.

The 6-oxa analogues of potent dopamine agonists, hexahydronaphthoxazines (4a,4b), have been tested for dopamine receptor binding and stimulating activity and were found to be almost inactive. pKa value determinations indicated that these compounds are protonated to approximately 2%, while potent compounds are protonated to a much greater extent. These results strongly support the assumption that the protonated form of DA agonists is the active species at the receptor.

Synthesis and evaluation of pharmacological and pharmacokinetic properties of monopropyl analogs of 5-, 7-, and 8-[[(trifluoromethyl)sulfonyl]oxy]-2-aminotetralins: central dopamine and serotonin receptor activity.

In order to explore further the structure-activity relationships of serotonergic and dopaminergic ligands, a series of enantiopure 5-, 7-, or 8-triflate (-OTf)-substituted 2-(monopropylamino)-tetralins have been synthesized and evaluated in in vitro binding and in vivo biochemical and behavioral assays in rats. Consequently, the 8-OTf-substituted compound R-(+)-6 was found to be a potent and selective 5-HT1A (5-hydroxytryptamine) receptor agonist inducing a full-blown 5-HT syndrome in normal rats, while the corresponding 5-OTf-substituted compound S-(-)-12 was found to be a preferential dopamine (DA) autoreceptor agonist. A partial 5-HT syndrome was also observed for S-(-)-12, while the corresponding R-(+)-12 was found to be inactive at the DA and 5-HT receptors both in vitro and in vivo. Compounds 6 and 12 were found to be major urinary metabolites following oral administration of their dipropyl analogs (2 and 13, respectively). Thus 6 was proposed to be the metabolite responsible for the full-blown 5-HT syndrome seen after oral (but not subcutaneous) administration of 2. Similarly, 12 was proposed to be the metabolite responsible for the partial 5-HT syndrome seen after oral (but not subcutaneous) administration of 13. The bioavailability of R-(+)-6 (7.6 +/- 1.1%) appeared to be slightly lower than that of 2 (11.2 +/- 5.2%), although the in vitro metabolism of R-(+)-6 appeared to be slower than that of 2. Therefore first-pass metabolism was not thought to be the reason for the lower bioavailability of R-(+)-6, as compared to 2.

Affinity for dopamine D2, D3, and D4 receptors of 2-aminotetralins. Relevance of D2 agonist binding for determination of receptor subtype selectivity.

A series of 2-aminotetralins, substituted with a methoxy or a hydroxy group on the 5- or 7-position, and with varying N-alkyl or N-arylalkyl substituents, were prepared and evaluated in binding assays for human dopamine (DA) D2, D3, and D4 receptors. Some members of this series were prepared in former studies, but were never tested in vitro with single receptor subtypes, and these were examined again. None of the tested 2-aminotetralins showed high affinity for the dopamine D4 receptor. However, a number of the 2-aminotetralins showed high affinity for both the D2 and the D3 DA receptors, as exemplified by compounds 11-15 and 21-26, while some had a reasonable selectivity for the DA D3 receptors. The affinities of the 2-aminotetralins for the D21, receptor depended on the type of radioligand (agonist or antagonist) used. The agonist affinity data, obtained by using the agonist ligand [3H]N-0437, are thought to be more relevant for calculating DA receptor subtype selectivity.

Synthesis and pharmacological evaluation of thiopyran analogues of the dopamine D3 receptor-selective agonist (4aR,10bR)-(+)-trans-3,4,4a,10b-tetrahydro-4-n-propyl-2H,5H [1]b enzopyrano[4,3-b]-1,4-oxazin-9-ol (PD 128907).

Benzopyranoxazine (+)-7 (PD 128907) is the most dopamine (DA) D3 receptor-selective agonist presently known. The only structural feature which distinguishes 7 from the analogous nonselective naphthoxazines is an oxygen atom in the 6-position. To extend this series of tricyclic DA agonists we used a classic bioisoster approach and synthesized thiopyran analogues of 7, which have a sulfur atom in the 6-position. We prepared trans-4-n-propyl-3,4,4a,10b-tetrahydro-2H,5H-[1]benzothiopyrano[4, 3-b]-1,4-oxazin-9-ol (9, trans-9-OH-PTBTO), its enantiomers ((+)-9 and (-)-9), the racemic cis-analogue (10), and the racemic trans-sulfoxide (11) and studied the potency and selectivity for DA receptors of these compounds. As with other rigid DA agonists, the highest affinity for DA receptors resided in one of the enantiomers, in this case the (-)-enantiomer of 9. On the basis of a single-crystal X-ray analysis of a key intermediate, the absolute configuration of (-)-9 was found to be 4aS,10bR, which is homochiral with (+)-(4aR,10bR)-7. In contrast to (+)-7 however, (-)-9 displayed no selectivity for any of the DA receptors. In addition, it has affinity for 5HT1A receptors. (+/-)-cis-4-n-Propyl-3,4,4a,10b-tetrahydro-2H,5H-[1]benzothiopyrano++ +[4,3-b]-1,4-oxazin-9-ol (10), which was expected to be inactive, displayed affinity and selectivity for the DA D3 receptor, whereas the sulfoxide 11 displayed some DA D3 selectivity, but with a lower affinity. Further pharmacological evaluation revealed that (-)-9 is a very potent full agonist at DA D2 receptors and a partial agonist at DA D3 receptors. The cis-analogue (+/-)-10 displayed the same profile, but with lower potency. These findings were confirmed in vivo: in reserpinized rats (-)-9 displayed short-acting activation of locomotor activity (DA D2 agonism) and also lower lip retraction and flat body posture, (5HT1A agonism). Compound (+/-)-10 had no effect on locomotor activity. In unilaterally 6-OH-DA lesioned rats, (-)-9 gave short-acting locomotor activation. Furthermore, in microdialysis studies in rat striatum, (-)-9 potently decreased DA release, confirming its activation of presynaptic DA D2 receptors.

Modification of behavioral effects of drugs in mice by neuroactive steroids.

RATIONALE: Neuroactive steroids represent a novel class of potential therapeutic agents (epilepsy, anxiety, migraine, drug dependence) thought to act through positive allosteric modulation of the GABA(A) receptor. A synthetically derived neuroactive steroid, ganaxolone (3alphahydroxy-3beta-methyl-5alpha-pregnan-20-one), is in phase-II clinical trials for epilepsy. Unlike traditional anticonvulsants such as diazepam and phenobarbital, ganaxolone shows equipotent suppression of both the seizure activity and the behavioral effects of pentylenetetrazol (PTZ) administration. OBJECTIVES: The present study explored possible reversal by ganaxolone and related neuroactive steroids of some behavioral effects of additional pharmacological challenges. METHODS: Direct behavioral observation and photocell-counted locomotor activity of male, Swiss-Webster mice were made with various compounds alone and in conjunction with ganaxolone. RESULTS: Ganaxolone both prevented and reversed PTZ-induced locomotor depression in mice. Further, ganaxolone reversed the locomotor depression induced by other convulsant/anxiogenic stimuli: bicuculline, picrotoxin and, to a lesser extent, yohimbine. Ganaxolone failed to reverse the locomotor stimulation induced by cocaine, methamphetamine, dizocilpine, and phencyclidine. In addition to ganaxolone, the endogenous neuroactive steroids allopregnanolone and pregnanolone and the synthetic neuroactive steroid Co 2-1068 also reversed observed behaviors and locomotor depression induced by PTZ. CONCLUSIONS: The present findings support the unique pharmacological effects of neuroactive steroids as a novel class of positive allosteric modulators of GABA.

Autoradiographic localisation of D3-dopamine receptors in the human brain using the selective D3-dopamine receptor agonist (+)-[3H]PD 128907.

The selective D3-dopamine receptor agonist 4aR, 10bR-(+)-trans-3,4,4a, 10b-tetrahydro-4-[N-propyl-2,3-3H]-2H,5H-[1] benzopyrano[4,3-b]-1,4-oxazin-9-ol ([3H]PD 128907) was used to visualise D3-dopamine receptors in whole hemisphere cryosections from post-mortem human brain. [3H]PD 128907 has an 18- to 40-fold selectivity for D3- over D2-dopamine receptors as compared to a 7- to 24-fold selectivity of the more commonly used ligand [3H]7-OH-DPAT. [3H]PD 128907 accumulated markedly in the nucleus accumbens and in the ventral parts of caudate nucleus and putamen, with a slightly heterogeneous (patch-matrix like) distribution. The binding in the lateral parts of caudate nucleus and putamen was much less dense. No binding was obtained in any other regions. A very high proportion of [3H]PD 128907 was specifically bound, as judged from the low binding remaining in the presence of the D2/D3-dopamine receptor antagonist raclopride. This gives the ligand a potential for the detection of low density D3-dopamine receptors in the human brain. The binding obtained with [3H]PD 128907 was qualitatively similar to that using [3H]7-OH-DPAT in the presence of GTP. However, [3H]7-OH-DPAT labelled, in contrast to [3H]PD 128907, also D3-dopamine receptors in neocortex. The new compound [3H]PD 128907 appears to be a suitable radioligand for autoradiographic examination of the D3-dopamine receptor localisation in the human brain, and should also be useful for pharmacological studies of this receptor subtype.

Characterization of the effect of dopamine D3 receptor stimulation on locomotion and striatal dopamine levels.

By examining the effect of dopamine (DA) D3 receptor stimulation on locomotor activity and extracellular levels of DA in striatum we show that inhibition of locomotor activity induced by DA D3 receptor-selective agonists is mediated by two interacting mechanisms: (1) directly via the stimulation of DA D3 receptors that inhibit locomotor activity, and (2) indirectly via a decrease in extracellular levels of DA. Thus, the moderately DA D3 receptor-selective agonist R-(+)-7-OH- DPAT (R-(+)-7-hydroxy-2-(N,N-di-n-propylamino)tetralin) decreased locomotor activity after administration of 10 nmol/kg and extracellular DA levels in accumbens and striatum after administration of 30 nmol/kg. A decrease in locomotor activity that coincided with a decrease in extracellular DA levels in striatum was observed after administration of 100 nmol/kg of the DA D3 receptor-selective agonist PD128907 ((+)-trans-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3 b]-1,4-oxasin-9-ol. In combination with the partial, DA D3 receptor-selective agonist PD151328 (2-[4[3-(4-phenyl)-1- piperazinyl)propoxy]phenyl]-benzamidazole), a reversal of the attenuating effect of PD128907 on locomotor activity was observed, without an effect on extracellular levels of DA. In combination with a low--10 nmol/kg--dose of haloperidol, a reversal of the inhibitory effect of PD128907 on locomotor activity was observed that coincided with an increase in extracellular levels of DA. In the presence of 0.5 mg/kg amphetamine, PD128907 decreased amphetamine-induced locomotor activity. This effect could be reversed by PD151328.

Is TL-99 a selective presynaptic dopamine receptor agonist?

The claim that the 2-aminotetralin analogue TL-99 is a selective presynaptic dopamine (DA) receptor agonist has been investigated both in vivo and in vitro in the rat. The pharmacological specificity of the hypomotility caused by TL-99 has been examined using various selective antagonists. In addition its effects on DA metabolism and noradrenaline (NA) and DA turnover (alpha-MT method) as well as its distribution in the brain have been studied. These in vivo studies provide evidence that although TL-99 is able to activate presynaptic DA receptors it is also a potent agonist of NA receptors as shown by the fact that the hypomotility could be partly reversed by the selective alpha 2-adrenoceptor antagonists yohimbine and piperoxan. Further supporting evidence for these findings was provided by in vitro studies on the inhibition of K+-induced [3H]dopamine, [14C]acetylcholine and [3H]noradrenaline release from striatal and cortical slices where it was shown that TL-99 is not only active at both pre- and postsynaptic DA receptors but also at alpha 2-NA receptors. For the latter receptor it had a potency comparable to that of the potent alpha 2-agonist clonidine and this may explain, to some extent, the hypomotility caused by TL-99. Thus, ascribing this hypomotility solely to an interaction with presynaptic DA receptors may be an oversimplification. It is therefore concluded that TL-99 should not be considered as a selective presynaptic DA receptor agonist.

Kinetic and pharmacological profiles of the in vitro binding of the potent dopamine agonist [3H]N,N-dipropyl-5,6-dihydroxy-2-aminotetralin to rat striatal membranes.

The in vitro binding of the new tritiated dopaminergic ligand [3H]N,N-dipropyl-5,6-dihydroxy-2-aminotetralin to rat striatal membranes is described. Binding assays were performed in 50 mM Tris-HCl pH 7.5 containing 1 mM EDTA and 0.01% ascorbic acid at 25 degrees C for 30 min. Specific binding at 0.5 nM [3H]DP-5,6-ADTN and 5 mg/ml membrane suspension was very high (87-93%) and was found to be linearly related with homogenate concentration over the range of 0.5-10 mg/ml (r = 0.9968). (+)Butaclamol 1 microM was used to define specific binding. Specific binding disappeared completely within 20 min when the tissue was incubated at 55 degrees C. Association and dissociation curves (obtained after addition of 1 microM (-)-DP-5,6-ADTN) were converted to linear logarithmic plots and the kinetic constants were computed (k1 = 0.054 min-1 and k-1 = 0.0188 min-1) as were the t1/2 for association (3.52 min) and dissociation (20.8 min). This resulted in an apparent KD of 0.34 nM. Increasing concentrations of [3H]DP-5,6-ADTN (0.05-3.0 nM) were found to saturate the receptor site. Linear transformation of the saturation curves and presentation of the curves as Eadie-Hofstee plots showed that only one set of receptors was labeled (nHill was 0.995 +/- 0.07) with a high affinity constant KD of 0.57 +/- 0.10 nM and a maximum number of binding sites Bmax of 18.6 +/- 2.4 pmol/g tissue. Various compounds were tested for their potency to displace the specific binding of [3H]DP-5,6-ADTN to striatal membranes (2.5 mg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

A prodrug of ADTN: selectivity of dopaminergic action and brain levels of ADTN.

The effects of administration of the prodrug dibenzoyl ADTN (DBADTN) on ADTN concentrations in rat brain and on behaviour in rats having a unilateral 6-hydroxydopamine lesion in the corpus striatum have been studied. Using a combination of HPLC and electrochemical detection as assay method it was found that there was a more selective accumulation of ADTN in the corpus striatum than in the cerebellum. In addition the accumulation of ADTN in the corpus striatum was slow in onset yet long in duration. The peak concentration of ADTN was relatively low and although it was sufficient to cause a strong stimulation of presynaptic DA receptors it did not cause significant rotation in the unilaterally lesioned rat. Prodrug methodology may thus prove useful in designing new selectively acting DA agonists.