Sarizotan, a serotonin 5-HT1A receptor agonist and dopamine receptor ligand. 1. Neurochemical profile.

Sarizotan exhibited high affinities only to serotonin 5-HT1A receptors and dopamine DA D4>D3>D2 receptors with the profile of a 5-HT1A agonist and DA antagonist demonstrated by the inhibition of cAMP-stimulation and guinea pig ileum contraction, decreased accumulation of the 5-HT precursor 5-hydroxytryptophan and increased levels of 5-HT metabolites, increased accumulation of DA precursor dihydroxyphenylalanine (DOPA) and the reduced levels of DA metabolites in intact rats. However, sarizotan at higher doses decreased DA precursor accumulation in reserpinized rats and induced contralateral rotational behavior in unilaterally substantia nigra lesioned rats, indicating some intrinsic dopaminergic activity; at D2 receptors sarizotan may act as a partial agonist, depending on the dopaminergic impulse flow. Sarizotan represents a new approach for the treatment of extrapyramidal motor complications such as l-DOPA-induced dyskinesia in Parkinson's disease.

Deammonification in biofilm systems: population structure and function.

For the development of alternative concepts for the cost effective treatment of wastewaters with high ammonium content and low C/N-ratio, autotrophic consortia of micro-organisms with the ability to convert ammonium directly into N2 are of particular interest. Several full-scale industrial biofilm plants eliminating nitrogen without carbon source for years in a stable process, are suspected for some time to harbor active anaerobic ammonium oxidizers in deeper, oxygen-limited biofilm layers. In order to identify the processes of the single-stage nitrogen elimination (deammonification) in biofilm systems and to allocate them to the responsible micro-organisms, a deammonifying moving-bed pilot plant was investigated in detail. 15N-labelled tracer compounds were used as well as 16S rDNA libraries and in situ identification of dominant organisms. The usage of rRNA-targeted oligonucleotide probes (FISH) was particularly emphasized on the ammonium oxidizers of the beta-subclass of Proteobacteria and on the members of the order Planctomycetales. The combined application of these methods led to a deeper insight into the population structure and function of a deammonifying biofilm.

Six years' practical experience with aerobic/anoxic deammonification in biofilm systems.

Nitrogen elimination through autotrophic micro-organisms is currently in the focus of research projects on the treatment of wastewater with high nitrogen contents, for instance to be able to develop and fix dimensioning parameters for purposeful application. In fact, several industrial plants have already shown for some years that the steady operation of nitrogen elimination without carbon demand is possible. Due to the low growth rates of the participating micro-organisms, these processes can be found in particular in biofilm systems, which also allow for the simultaneous running of the two basic processes. In the following, we will discuss on the basis of the operation results of industrial and pilot-scale plants the operation stability of the aerobic/anoxic deammonification, and explain which experiences are available in particular for conversion in biofilm systems.

Single stage biological nitrogen removal by nitritation and anaerobic ammonium oxidation in biofilm systems.

In full scale wastewater treatment plants with at times considerable deficits in the nitrogen balances, it could hitherto not be sufficiently explained which reactions are the cause of the nitrogen losses and which micro-organisms participate in the process. The single stage conversion of ammonium into gaseous end-products--which is henceforth referred to as deammonification--occurs particularly frequently in biofilm systems. In the meantime, one has succeeded to establish the deammonification processes in a continuous flow moving-bed pilot plant. In batch tests with the biofilm covered carriers, it was possible for the first time to examine the nitrogen conversion at the intact biofilm. Depending on the dissolved oxygen (DO) concentration, two autotrophic nitrogen converting reactions in the biofilm could be proven: one nitritation process under aerobic conditions and one anaerobic ammonium oxidation. With the anaerobic ammonium oxidation, ammonium as electron donor was converted with nitrite as electron acceptor. The end-product of this reaction was N2. Ammonium and nitrite did react in a stoichiometrical ratio of 1:1.37, a ratio which has in the very same dimension been described for the ANAMMOX-process (1:1.31 +/- 0.06). Via the oxygen concentration in the surrounding medium, it was possible to control the ratio of nitritation and anaerobic ammonium oxidation in the nitrogen conversion of the biofilm. Both processes were evenly balanced at a DO concentration of 0.7 mg/l, so that it was possible to achieve a direct, almost complete elimination of ammonium without addition of nitrite. One part of the provided ammonium did participate in the nitritation, the other in the anaerobic ammonium oxidation. Through the aerobic ammonium oxidation into nitrite within the outer oxygen supplied layers of the biofilm, the reaction partner was produced for the anaerobic ammonium oxidation within the inner layers of the biofilm.

5-HT reuptake inhibitors with 5-HT(1B/1D) antagonistic activity: a new approach toward efficient antidepressants.

As part of our research program toward new, potential antidepressants, a series of unsymmetrical ureas has been prepared and evaluated as 5-HT reuptake inhibitors with 5-HT(1B/1D) antagonistic activities. The design of these compounds was based on coupling of various indole derivatives, previously shown to inhibit 5-HT reuptake, to three different aniline moieties, which are part of known 5-HT(1B/1D) ligands. Binding experiments in rat frontal cortex using [(125)I]iodocyanopindolol, in calf striatum using [(3)H]5-HT, and in rat hippocampus using [(3)H]8-OH-DPAT as radioligands, respectively, revealed significantly higher affinity at the 5-HT(1B) receptor as compared to the affinities for the 5-HT(1A) and 5-HT(1D) receptors for a number of compounds, among them 4-(5-fluoro-1H-indol-3-yl)piperidine-1-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (5), the corresponding 4-fluoro-1H-indol-3-yl analogue 21a, and the corresponding 6-fluoro-1H-indol-3-yl analogue 21b. Conformational restriction of the aniline moiety in 5 only slightly enhanced the 5-HT(1B) affinity, whereas introduction of an aniline moiety with higher conformational flexibility resulted in a less potent 5-HT(1B) receptor ligand as compared to 5. The functional 5-HT(1B/1D) antagonistic activity was investigated using the rabbit saphenous vein model as well as the [(3)H]5-HT release from guinea pig cortical slices. All new compounds tested in the rabbit saphenous vein model were shown to antagonize the sumatriptan-evoked contractile responses with pA(2) values ranging from 7.3 to 8.7. These observations were consistent with the results of the cortical slice model, in which the ureas were found to block the sumatriptan-induced inhibition of potassium-evoked [(3)H]5-HT release. The 5-HT reuptake inhibition of the ureas determined in rat brain synaptosomes was found to be either increased or decreased as compared to the uncoupled indole derivatives indicating that the reuptake inhibition shown by the ureas is not only due to the indole part but also affected by the aniline moiety of the molecule. Among this series of compounds described the ureas 5, 21a, and 21b seem to be the most interesting candidates showing both 5-HT reuptake inhibition and 5-HT(1B/1D) antagonism in vitro. This dual pharmacological profile should in theory lead to a pronounced enhancement in serotonergic neurotransmission and consequently to a more efficient treatment of depression.

New (sulfonyloxy)piperazinyldibenzazepines as potential atypical antipsychotics: chemistry and pharmacological evaluation.

A series of 2- or 8-trifluoromethylsulfonyloxy (TfO) and 2- or 8-methylsulfonyloxy (MsO) 11-piperazinyldibenzodiazepines, -oxazepines, and -thiazepines were synthesized and evaluated in pharmacological models for their potential clozapine-like properties. In receptor binding assays, the 2-TfO analogues (18a, GMC2-83; 24, GMC3-06; and previously reported GMC1-169, 9a) of the dibenzazepines have profiles comparable to that of clozapine, acting on a variety of CNS receptors except they lack M1 receptor affinity. Introduction of 2-TfO to clozapine leads to compound 9e (GMC61-39) which has a similar binding profile as that of clozapine including having M1 receptor affinity. Interestingly, the MsO analogues, as well as the 8-TfO analogues, have no or weak dopaminergic and serotonergic affinities, but all 8-sulfonyloxy analogues do have M1 affinities. In behavioral studies performed to indicate the potential antipsychotic efficacy and the propensity to induce EPS, 2-TfO analogues blocked effectively the apomorphine-induced climbing in mice in a dose-dependent manner with ED50 values (mg/kg) of 2.1 sc for 9a, 1.3 po for 18a, 2.6 sc for 24, and 8.2 sc for 9e. On the other hand, they showed a clear dose separation with regard to their ED50 values (mg/kg) for indicating catalepsy in rats (>44 sc for 9a, 28 po for 18a, 30 sc for 24, and >50 sc for 9e, respectively), thus implicating a more favorable therapeutic ratio (K/A, ED50 climbing/ED50 catalepsy) in comparison with typical neuroleptics such as haloperidol and isoclozapine. Furthermore, compound 18a was also demonstrated to be an orally potent DA antagonist with an ED50 value of 0.7 mg/kg po in the ex vivo L-DOPA accumulation model. The present study contributes to the SAR of 11-piperazinyldibenzazepines, and the 2-TfO analogues of 11-piperazinyldibenzazepines are promising candidates as clozapine-like atypical antipsychotics with low propensity to induce EPS.

Effects of GR-89696 and the novel peripherally selective OP2 agonists, EMD-61569 and EMD-61747, against focal cerebral ischemia in the rat.

We examined whether increases in blood-brain barrier (BBB) permeability occurring after stroke can be exploited to apply protective substances selectively to ischemic tissue. To do this, the actions of the peripherally selective OP2 agonists, EMD-61569 and EMD-61747, have been compared with those of the centrally acting OP2 agonist, GR-89696, in the rat permanent focal ischemia model. EMD-61569, EMD-61747 and GR-89696 all bound with high affinity to OP2 receptors and were potent agonists in the rabbit vas deferens functional assay. These substances also potently inhibited electrically-induced overflow of dopamine from slices of rat nucleus accumbens. EMD-61747 and EMD-61569 penetrate poorly into the CNS under normal conditions and reverse haloperidol-induced L-DOPA accumulation in the nucleus accumbens of the rat only at high doses, in contrast to GR-89696. Permanent unilateral occlusion of the middle cerebral artery (MCAO) was associated with a disruption of the BBB and an increase in the concentration of EMD-61747 in the area of the infarct. GR-89696 at a dose of 0.1 mg/kg s.c. produced a reduction in infarct volume by 38% after MCAO, EMD-61569 and EMD-61747 had no influence on swelling and ischemic damage. We conclude that EMD-61747 and EMD-61569 are potent OP2 agonists, which usually have a limited ability to penetrate the BBB. The change in the properties of the BBB in ischemic tissue was not sufficient to elicit neuroprotection, since both EMD-61747 and EMD-61569 were inactive in the focal ischemia model. Conversely, GR-89696 had a robust protective action, and probably powerful OP2-typical side effects as a consequence of its unrestricted central activity.

Effects of alpha-phenyl-tert-butylnitrone and selegiline on hydroxyl free radicals in rat striatum produced by local application of glutamate.

The hydroxyl radical is a very reactive oxygen species that damages biomolecules in the brain and in other tissues. The possible pharmacological intervention to prevent hydroxyl radical formation was studied in vivo using the microdialysis technique in brains of nonanesthetized rats. Hydroxyl radicals form stable adducts [mainly 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-DHBA)] via an aromatic hydroxylation reaction with salicylic acid. 2,3-DHBA was separated and quantified by HPLC and electrochemical detection. Microdialysis probes were implanted into the striatum 1 day before measurement of levels of hydroxyl radicals. The next day, the probes were first perfused for 120 min with a modified Ringer's solution containing 5 mM salicylic acid, to obtain stable baselines. Afterward, the perfusion solution was switched to another solution that in addition contained 50 mM glutamate, to stimulate radical formation. Twenty minutes later, alpha-phenyl-tert-butylnitrone (PBN; 100 mg/kg), selegiline (10 mg/kg), or saline was administered intraperitoneally. The glutamate perfusion produced marked two- to 2.5-fold increases in 2,3-DHBA content. Treatment with PBN significantly antagonized the rise of 2,3-DHBA level, indicating that PBN is a direct radical scavenger not only in vitro but also in vivo. Acute treatment with selegiline failed to reduce significantly the glutamate-induced radical formation. The acute experiments presented here do not support the suggestion that the neuroprotective effects of selegiline described in the literature are due to a potential hydroxyl radical scavenging property of the drug.

SAR of novel biarylmethylamine dopamine D4 receptor ligands.

SAR for a novel series of dopamine D4 receptor ligands is shown. Very selective, highly potent compounds like 1-(2-pyrimidinyl)-4-(3-(3-thienyl)-benzyl)-piperazine (5f) and 2-(4-(1-fluorenylmethyl)-1-piperazinyl)-pyrimidine (8c) were obtained.

The peripherally acting kappa-opiate agonist EMD 61753 and analogues: opioid activity versus peripheral selectivity.

EMD 61753 (N-methyl-N-[(1S)-1-phenyl-2-((3S)-3-hydroxypyrrolidin-1-yl) -ethyl ]- 2,2-diphenyl-acetamide hydrochloride) is a peripherally selective kappa-opiate agonist. It exhibits antihyperalgesic activity in animal models of inflammatory pain at doses which do not cause signs of central action. The structure of this compound was varied in different ways and the resulting derivatives were tested for affinity to the kappa-receptor. Furthermore, those compounds with binding values comparable to that of EMD 61753 were tested for central activity. This was done by measuring the extent to which the haloperidol-induced L-DOPA accumulation in the nucleus accumbens of the rat could be reversed after application of 10 mg/kg s.c. of the test compound. Structure-activity relationships revealed that none of the analogues or reference compounds tested is superior to the parent compound with regard to its favourable ratio between kappa-receptor affinity and peripheral selectivity.

Studies on the interaction of roxindole with brain monoamine oxidases and dopaminergic neurones in vitro and in vivo.

Roxindole, a structurally novel psychotropic indolylbutyl-4-phenyltetrahydropyridine, was studied with respect to the formation of potentially neurotoxic pyridinium metabolites in comparison to MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). In contrast to MPTP, roxindole failed to serve as a substrate for monoamine oxidases (MAO) from mouse, monkey and human brain in vitro. Accordingly, neither the putative MAO-oxidation product of roxindole (ROX+) nor MPP+ (1-methyl-4-phenylpyridinium ion) was detected in mouse striatum after high subcutaneous doses of roxindole in spite of the presence of approximately 6 micrograms of roxindole per g of striatum in these animals. After multiple subcutaneous treatments with 95.2 mg/kg roxindole, no long-term striatal dopamine depletions were observed in contrast to MPTP. Furthermore, unlike MPP+, ROX+ did not induce release of previously accumulated 3H-dopamine in mouse striatal slices indicating that ROX+ cannot utilize the dopamine uptake carrier to enter neurones. ROX+ at doses up to 100 mg/kg subcutaneously failed to alter striatal biogenic amine levels and gross behaviour of mice. Thus, no MPTP-like neurotoxic metabolites are formed from roxindole in vivo and neurotoxic effects of ROX+, even if formed in minute amounts by some MAO-independent pathway, are highly unlikely.

Sensitivity of dopamine D2 receptors following long-term treatment with roxindole.

The selective presynaptic dopamine D2 receptor agonist roxindole was studied in specific pre- and postsynaptic models in rats to see whether it induced changes in dopamine D2 receptor sensitivity. Following treatment with 0.3 or 3 mg/kg per day i.p. for 21 days, the reversal of gamma-butyrolactone-induced striatal dihydroxyphenylalanine accumulation was unchanged as compared to that after acute treatment. The efficacy of roxindole in this model was not decreased after long-term treatment. Likewise, treatment for 19 days with up to 10 mg/kg per day i.p. failed to induce behavioral supersensitivity, i.e. potentiation of apomorphine-induced stereotypies. In a cotreatment paradigm with haloperidol (1 mg/kg per day p.o.), roxindole (10mg/kg per day i.p.) did not alter the behavioral supersensitivity measured after a drug washout phase as compared to the effect of haloperidol alone; however, stereotypies were observed after termination of haloperidol but continuation of roxindole treatment. In contrast, roxindole (10 mg/kg i.p.) induced only weak stereotypies in haloperidol-sensitized rats when given after the washout phase instead of apomorphine. The results indicate that roxindole induces neither desensitization of presynaptic nor supersensitization of postsynaptic dopamine D2 receptors. Nevertheless, in add-on clinical studies with neuroleptics, switching of treatment regimens should be performed gradually over several days until further experience is available.

Central and peripheral actions of the novel kappa-opioid receptor agonist, EMD 60400.

1. The pharmacological characteristics of the kappa-opioid receptor agonist, EMD 60400, have been investigated, with particular reference to its central and peripheral sites of action and its ability to influence nociception. The kappa agonists ICI 197067 and ICI 204448 were tested for purposes of comparison. 2. EMD 60400 and ICI 197067 bind with high affinity (IC50 values of 2.8 and 1.5 nM, respectively) and high selectivity to kappa-opioid receptors. ICI 204448 has a lower binding affinity (IC50 13.0 nM) and selectivity for kappa-opioid receptors. 3. EMD 60400, ICI 197067, and ICI 204448 are full and potent agonists in the rabbit vas deferens in vitro assay for kappa-opioid receptors (IC50 values of 41.8, 15.7 and 15 nM, respectively). 4. Ex vivo binding experiments in mice revealed that EMD 60400 and ICI 197067 were well taken up after s.c. administration. Brain levels of EMD 60400 were lower than those of ICI 197067 at comparable doses, indicating that EMD 60400 does not penetrate into the CNS as well as ICI 197067. 5. Haloperidol-induced DOPA accumulation in the nucleus accumbens of the rat was dose-dependently reversed by s.c. application of EMD 60400 and ICI 197067 at doses of and above 3 and 0.3 mg kg-1, respectively. ICI 204448 had no effect on DOPA accumulation at 30 mg kg-1, s.c. 6. Prolongation of hexobarbitone-induced sleeping time in mice and motor impairment in the rat rotarod test were observed for EMD 60400 at doses above 3 and 2.5 mg kg-1, s.c., respectively, and for ICI 197067 at doses above 0.3 and 0.25 mg kg-1, s.c., respectively. ICI 204448 was inactive in these tests at doses of 30 and 100 mg kg-1, s.c., respectively.7. EMD 60400 applied s.c. produced dose-dependent naloxone-reversible antinociception in the mouse formalin test (1st and 2nd phase ID50 0.44 and 0.47 mg kg-1, respectively) and rodent writhing test (ID50 mouse 0.55 mg kg-1 and rat 0.3mg kg-1). Furthermore, EMD 60400 was considerably more potent in the rat pressure pain test after the induction of inflammation with carrageenin than under normalgesic conditions (ID50 values 0.1 Microg kg-1 and 4.0 mg kg-1, s.c., respectively). The action of EMD 60400 (50 microgkg-1, s.c.) in the hyperalgesic pressure pain test was completely antagonized by injection of the K-opioid antagonist, norbinaltorphimine (100 microg) into the inflamed tissue, thus demonstrating the peripheral opioid nature of this effect.8. EMD 60400 produced dose-dependent inhibition of neurogenic plasma extravasation elicited byantidromic electrical stimulation of the rat saphenous nerve (ID50 value 0.3 mg kg-1, i.v.). This inhibition was completely antagonized by intraplantar injection of norbinaltorphimine (50 microg).9. EMD 60400, ICI 197067, and ICI 204448 have diuretic effects in rats at doses of and above 0.1, 0.01,and 0.3 mg kg-1, s.c., respectively. An antidiuretic action was also observed with ICI 197067 at very low doses (3 and 6 microgkg-1, s.c.).10. Pharmacological and biochemical data therefore indicate that the three K-opioid receptor agonists tested here have different tendencies to elicit centrally-mediated sedation and putative aversion(ICI 197067 > EMD 60400 > ICI 204448) which correspond to their ability to cross the blood-brain barrier. EMD 60400 combines high affinity and selectivity for the K receptor with a degree of peripheral selectivity. The peripheral actions of systemically-applied EMD 60400 against hyperalgesic pressure pain and neurogenic inflammation are very probably mediated by opioid receptors on the endings of sensory nerve fibres.

Human, mouse, and rat calnexin cDNA cloning: identification of potential calcium binding motifs and gene localization to human chromosome 5.

Calnexin is a 90-kDa integral membrane protein of the endoplasmic reticulum (ER). Calnexin binds Ca2+ and may function as a chaperone in the transition of proteins from the ER to the outer cellular membrane. We have purified human calnexin in association with the human interferon-gamma receptor and cloned calnexin cDNA from placenta. Fragments of calnexin have been prepared as glutathione S-transferase fusion proteins and analyzed for their abilities to bind 45Ca2+ and ruthenium red. A subdomain containing four internal repeats binds Ca2+ with the highest affinity. This sequence is highly conserved when compared to calreticulin (a luminal ER protein), an Onchocerca surface antigen, and yeast and plant calnexin homologues. Consequently, this sequence represents a conserved motif for the high-affinity binding of Ca2+, which is clearly distinct from the "E-F hand" motif. An adjacent subdomain, also highly conserved and containing four internal repeats, fails to bind Ca2+. The carboxyl-terminal, cytosolic domain is highly charged and binds Ca2+ with moderate affinity, presumably by electrostatic interactions. The calnexin amino-terminal domain (residues 1-253) also binds Ca2+, in contrast to the amino-terminal domain of calreticulin, which is relatively less acidic. We have also determined the cDNA sequences of mouse and rat calnexins. Comparison of the known mammalian calnexin sequences reveals very high conservation of sequence identity (93-98%), suggesting that calnexin performs important cellular functions. The gene for human calnexin is located on the distal end of the long arm of human chromosome 5, at 5q35.

Synthesis and dopaminergic activity of some 3-(1,2,3,6-tetrahydro-1-pyridylalkyl)indoles. A novel conformational model to explain structure-activity relationships.

The synthesis and dopaminergic properties of a novel type of dopamine agonist is described. The number and kind of essential structural elements differ significantly from that of the rigid apomorphine-type dopamine agonists. Using standard molecular modeling techniques, a conformational model is developed proposing a U-shaped conformation which might be energetically preferred through aromatic pi-pi-interactions between both of the electron rich aromatic structural elements of this class of compounds. Superimposition of conformations of the lead compound 28 with apomorphine yields a novel model explaining the atypical structure-activity relationships found in this class of indolealkylamines.

Pharmacological basis for antihypertensive therapy with a novel dopamine agonist.

In the past, nearly all major mechanisms involved in the regulation of blood pressure have become targets of antihypertensive drugs. They include the brain stem with its neuronal circuits of central cardiovascular regulation, the sympathetic neuro-effector system, the kidney, the renin angiotensin aldosterone system and the vascular smooth muscle cell. There are various ways of influencing the function of the sympathetic nervous system, but the clinical potential of one mechanism of action has not yet been explored in detail. Drugs that inhibit noradrenaline release through stimulation of inhibitory receptors located at adrenergic nerve terminals in the cardiovascular system (inhibitory presynaptic receptors) are not available for the treatment of hypertension. Among the multiple presynaptic receptors, dopamine receptors which belong to the dopamine2 subtype, are of particular interest. Carmoxirole is a novel indole derivative with a potent agonist action selective for dopamine2-receptors of the periphery. Experimental evidence shows that carmoxirole lowers blood pressure in various models of hypertension mainly or exclusively through inhibition of noradrenaline release from sympathetic nerve endings. This effect of carmoxirole is mediated by presynaptic dopamine receptors with the characteristic that release inhibition is restricted to low rates of sympathetic nerve discharge.

The human platelet-activating factor receptor gene (PTAFR) contains no introns and maps to chromosome 1.

Platelet-activating factor (PAF), a phospholipid, exhibits a variety of potent inflammatory bioactivities that are mediated by a specific cell surface receptor. The gene for the human PAF receptor (PTAFR) has been isolated by hybridization with a guinea pig probe. The coding sequence contains no intervening sequences. The encoded protein is highly homologous to the guinea pig PAF receptor (82% identity) and contains seven putative transmembrane domains. The PAF receptor therefore appears to be a member of the G protein coupled family of receptors and exhibits significant similarity to many members of the family. Analysis of somatic cell hybrids suggests that the PAF receptor is encoded by a single gene on human chromosome 1.

Neurochemical profile of EMD 45609 (carmoxirole), a dopamine DA2-receptor agonist.

A new potential antihypertensive drug, EMD 45609 (carmoxirole), has been characterized in various in vivo and in vitro models. EMD 45609 displayed high affinity for dopamine D2-receptors combined with negligible binding to D1-receptors in binding assays in vitro. However, in tests in vivo for central D2-receptor activity, EMD 45609 exhibited only weak activity. Thus, after p.o. administration, striatal L-DOPA accumulation in intact rats was unchanged up to 100 mg/kg p.o., i.e. doses 100 times higher than those reported to induce depressor activity. Central dopamine agonistic activity could only be verified in the more sensitive model of the reserpinized rat. EMD 45609 was more than 30 times less potent, however, than LY 141865 in reserpinized rats after s.c. administration. Similarly, in rats with 6-hydroxydopamine induced unilateral lesions of the substantia nigra, EMD 45609 was only marginally active. The shallow dose response curves and the submaximal effects obtained for central dopaminergic activity, as reflected in the inhibition of striatal L-DOPA accumulation, suggest that EMD 45609 is a partial dopamine D2-receptor agonist and in addition, owing to its ionizable structure, passes less readily into the brain than several reference compounds. A marked affinity was found towards 5-HT1A-receptors in vitro, whereas affinity for alpha 1- and alpha 2-adrenoceptors was low; accordingly, central alpha 2-adrenoceptor activity was not detected as EMD 45609 failed to affect hypothalamic L-DOPA accumulation even at 100 mg/kg s.c. In accordance with its high affinity for D2-receptors in vitro, EMD 45609 inhibited field stimulated noradrenaline release from rabbit ear arteries in nanomolar threshold concentrations at 0.5 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)

Allelic polymorphism in transcriptional regulatory regions of HLA-DQB genes.

Class II genes of the human major histocompatibility complex (MHC) are highly polymorphic. Allelic variation of structural genes provides diversity in immune cell interactions, contributing to the formation of the T cell repertoire and to susceptibility to certain autoimmune diseases. We now report that allelic polymorphism also exists in the promoter and upstream regulatory regions (URR) of human histocompatibility leukocyte antigen (HLA) class II genes. Nucleotide sequencing of these regulatory regions of seven alleles of the DQB locus reveals a number of allele-specific polymorphisms, some of which lie in functionally critical consensus regions thought to be highly conserved in class II promoters. These sequence differences also correspond to allelic differences in binding of nuclear proteins to the URR. Fragments of the URR of two DQB alleles were analyzed for binding to nuclear proteins extracted from human B lymphoblastoid cell lines (B-LCL). Gel retardation assays showed substantially different banding patterns to the two promoters, including prominent variation in nuclear protein binding to the partially conserved X box regions and a novel upstream polymorphic sequence element. Comparison of these two polymorphic alleles in a transient expression system demonstrated a marked difference in their promoter strengths determined by relative abilities to initiate transcription of the chloramphenicol acetyltransferase reporter gene in human B-LCL. Shuttling of URR sequences between alleles showed that functional variation corresponded to both the X box and upstream sequence polymorphic sites. These findings identify an important source of MHC class II diversity, and suggest the possibility that such regulatory region polymorphisms may confer allelic differences in expression, inducibility, and/or tissue specificity of class II molecules.

Total versus unspecific binding: standardization and optimization of receptor-ligand binding assays.

Intra- and interassay imprecision was evaluated in 3 in vitro ligand binding assays using different types of filtering devices, glass fiber filters and variations in methodology. In the [3H]-spiroperidol test, the already low unspecific binding seemed to be dependent on the type of filters employed, whereas in the [3H]-ketanserin- and [3H]-GR 65630-binding tests, differences were within the range of the normal variabilities. However, in the latter test, which is problematic owing to the high unspecific binding of [3H]-GR 65630, it was found that although the percentage of specific binding was fairly constant on different sheets, large differences in the absolute amounts of total and unspecific binding were observed on consecutive sheets in the same experiment. Thus, it is critically important to filter samples for total and unspecific binding together on the same filter sheet for the calculation of specific binding. Under these precautions, highly reproducible results for IC50-values in the screening of potential 5HT3-receptor ligands were obtained in spite of using rat entorhinal cortex, a relatively large area with suboptimal 5HT3-receptor density. In contrast, when using rat area postrema, which is optimal with respect to receptor density, more than 10 times the number of rats is necessary for a competition experiment due to the small size of this brain part. Since IC50-values for both areas compare favorably, entorhinal cortex should be used for ethical reasons and to minimize costs.