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A better understanding of Continuous Positive Airway Pressure (CPAP) adherence is a priority in improving patient care. To Identify adherence typology with a longitudinal approach, and explore the early determinants of lower adherence to CPAP. Obstructive sleep apnea patients ( N = 204). Prospective and longitudinal study.A classification into four profiles was observed: “ Regular Adherents,” “ Non-Regular Adherents,” “ Persistent Non-Adherents,” and “ Non-Persistent Non-Adherents.” Specific biopsychosocial factors make it possible to evaluate the risk of belonging to a lower adherence profile. We propose a novel approach of CPAP treatment adherence. Several pre-determinants have been identified.

The Innovative Approaches in Anti-Cancer Monoclonal Antibodies meeting, held on March 20, 2012 in Lyon, was organized by Canceropole Lyon Auvergne-Rhone-Alps in partnership with the French competitiveness cluster Lyonbiopole. CLARA is one of the seven cancer research clusters within France in charge of facilitating Translational Oncology Research by taking into account the objectives of the French National Cancer Plans I and II and, in coordination with the French National Cancer Institute and local authorities (mainly Grand Lyon, Rhone County and Rhone-Alpes Region), to perform economic development of research findings. The contribution of lectures by outstanding speakers as described in this report, the organization of two-round tables: “Antibody treatment in cancer: Unmet needs in solid tumors and hematological malignancies,” and “From chimeric to more than human antibodies,” together with face-to-face meetings, was shared by over 230 participants. The lectures provided an overview of the commercial pi...

1 Two novel selective 5-HT6 receptor ligands E-6801 (6-chloro-N-(3-(2-(dimethylamino)ethyl)-1H-indol-5-yl)imidazo[2,1-b]thiazole-5-sulfonamide) and E-6837 (5-chloro-N-(3-(2-(dimethylamino)ethyl)-1H-indol-5-yl)naphthalene-2-sulfonamide) were investigated and compared to the putative 5-HT6 receptor antagonists SB-271046 (5-chloro-N-(4-methoxy-3-(piperazin-1-yl)phenyl)-3-methylbenzo[b]thiophene-2-sulfonamide) and Ro 04-06790 (N-(2,6-bis(methylamino)pyrimidin-4-yl)-4-aminobenzenesulfonamide) using a cAMP-mediated pathway. 2 Forskolin stimulation, to increase the magnitude of agonist cAMP responses, and site-directed mutagenesis of the 5-HT6 receptor, in order to yield constitutively active receptor, were applied. 3 5-HT (Emax, % over basal: 200), E-6801 (120) and E-6837 (23) induced cAMP formation at the rat 5-HT6 receptor. In the copresence of forskolin, cAMP responses were more potent and enhanced to 294 (5-HT, % over forskolin), 250 (E-6801) and 207 (E-6837), respectively. 5-HT-mediated cAMP formation was dose-dependently blocked by SB-271046 (pA2: 8.76±0.22) and Ro 04-6790 (pA2: 7.89±0.10) and not affected by the copresence of forskolin. Both E-6801 and E-6837 yielded partial antagonism of the 5-HT response in the absence of forskolin, whereas antagonism was either completely absent (E-6801) or attenuated (E-6837) in the copresence of forskolin. Intrinsic activity of these 5-HT6 receptor ligands at a constitutively active human S267K 5-HT6 receptor in Cos-7 cells indicated similar efficacy (Emax, % over basal) for 5-HT (97), E-6801 (91) and E-6837 (100), while Ro 04-6790 (-33) and SB-271046 (-39) were equi-efficacious inverse agonists. 4 The use of either forskolin or a constitutively active S267K 5-HT6 receptor enhances the resolution for monitoring the efficacy of 5-HT6 receptor ligands. E-6801 and E-6837 are potent partial agonists at the 5-HT6 receptor. Ro 04-6790 and SB-271046 appear to act as inverse agonists/antagonists. British Journal of Pharmacology (2006) 148, 1133–1143. doi:10.1038/sj.bjp.0706827

We hypothesized that the limited acute therapeutic effectiveness of tryptamine derivatives in alleviating migraine headache could be explained by the relatively low intrinsic activity of these agents at 5-HT1B/1D receptors. Donitriptan is a novel arylpiperazide 5-hydroxytryptamine (5-HT) derivative which was designed to exploit the higher potency and efficacy properties of 5-HT compared to tryptamine at 5-HT1B/1D receptors. In vitro, donitriptan has subnanomolar affinity for nonhuman and human 5-HT1B/1D receptors and micromolar affinity for the 5-HTip subtype. Donitritpan potently inhibited forskolin-induced cAMP formation and enhanced specific GTP35γS specific binding to a greater extent than tryptamine derivatives and equivalent to 5-HT in C6 cells expressing human 5-HT1B or 5-HT1D receptors. Donitriptan produced more potent and larger amplitude increases in hyperpolarizing Ca2+-dependent K+ current than sumatriptan in guinea pig isolated trigeminal ganglion neurons, and was more potent than tryptamine derivatives in eliciting contractile responses in rabbit isolated saphenous vein rings. In vivo, donitriptan evoked more potent, longer-lasting and greater amplitude carotid vasoconstrictor responses than tryptamine derivatives in anesthetized pigs; and in contrast to sumatriptan, naratriptan or zolmitriptan, produced long-lasting, dose-dependent decreases in unilateral carotid blood flow in conscious dogs at doses from 0.63 mg/kg p.o. without affecting heart rate or behavior. Oral donitriptan also evoked hypothermic responses in guinea pigs suggesting that the compound gains access to the brain. Donitriptan is thus a selective, potent 5-HT1B/1D receptor agonist which can be distinguished from tryptamine derivatives in consistently exerting high intrinsic activity at these receptors in a series of vascular and neuronal models relevant to migraine. Advantages in terms of therapeutic effectiveness in the acute relief of migraine headache over currently available triptans can be expected to include greater response rates and consistency of pain relief, a lower incidence of migraine recurrence and better tolerability. The acute anti-migraine potential of the first high efficacy 5-HT1B/1D agonist of its kind, donitriptan, is currently being investigated in man.

Neuroleptic drugs have been suggested to act as inverse agonists at the dopamine D2 receptor. Nevertheless, the capacity with which inverse agonism at this receptor subtype can be resolved is limited. Modulation of the constitutive activation of the D2 receptor was investigated in different cellular systems by monitoring either [35S]GTPγS binding responses at mutant Thr343Ser D2short receptor or inositol phosphates formation mediated by a chimeric D2/α1B 3ICL receptor. A weak (about −20% vs. basal [35S]GTPγS binding response) inverse agonist activity of putative dopamine antagonists (i.e., nemonapride, haloperidol or (+)-butaclamol) was observed with digitonin-permeabilized Chinese hamster ovary (CHO)-K1 cells stably expressing a mutant Thr343Ser D2short receptor only if a high (150 mM) KCl concentration was present in the binding buffer. No ligand-mediated decrease in basal [35S]GTPγS binding was observed on membrane preparations of the same cells. Markedly increased inverse agonist responses were obtained with a series of dopamine antagonists by exchange of the D2short receptor's 3ICL by that of the α1B-adrenoceptor and incorporation of an activating mutation (Ala279Glu) in the distal BBXXB motif of its 3ICL and by co-expression with a Gα11 protein. This chimeric D2/α1B receptor construct displayed a ligand binding profile comparable to that of the wt D2short receptor and an effector activation profile close to that of the wt α1B-adrenoceptor. Most of the putative dopamine antagonists attenuated by −54% to −59% basal inositol phosphates (IP) formation, thus clearly acting as inverse agonists. Ziprasidone behaved virtually as a silent antagonist (+5% vs. basal IP level) and antagonised both dopamine (pKB: 7.61)- and tropapride (pKB: 8.52)-mediated IP responses. Clozapine, olanzapine and raclopride displayed partial inverse agonist properties (−31%, −67% and −71% vs. tropapride 1 μM, respectively), whereas bromerguride (+63%) and (+)-UH 232 (+88%) demonstrated positive agonism.

A dopamine D(2Short) receptor:G(alphao) fusion protein was expressed in Sf9 cells using the baculovirus expression system. [(3)H]Spiperone bound to D(2Short):G(alphao) with a pK(d) approximately 10. Dopamine stimulated the binding of [(35)S]guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) to D(2Short):G(alphao) expressed with Gbeta(1)gamma(2) (E(max)>460%; pEC(50) 5.43+/-0.06). Most of the putative D(2) antagonists behaved as inverse agonists (suppressing basal [(35)S]GTPgammaS binding) at D(2Short):G(alphao)/Gbeta(1)gamma(2) although (-)-sulpiride and ziprasidone were neutral antagonists. Competition of [(3)H]spiperone binding by dopamine and 10,11-dihydroxy-N-n-propylnorapomorphine revealed two binding sites of different affinities, even in the presence of GTP (100 micro M). The D(2Short):G(alphao) fusion protein is therefore a good model for characterising D(2) receptors.

Whereas agonist-directed differential signaling at a single receptor subtype has become an accepted pharmacological concept, distinct behaviors by ligands that are assumed to be antagonists is less documented. The intrinsic activity and capacity of antagonism for a new series of imidazoline-derived adrenergic ligands analogous to dexefaroxan were investigated by measuring two distinct signaling pathways at the recombinant human alpha 2A-adrenoceptor (alpha 2A AR): 1) pertussis toxin-resistant guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding responses mediated by either a recombinant G alpha oCys351Ile or G alpha i2Cys352Ile protein in CHO-K1 cells, and 2) inhibition of cAMP formation in a stably transfected C6-glial cell line. Ligands could be differentiated as inverse agonists [i.e., 2-(4-methoxy-2-ethyl-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 851062], neutral antagonists [i.e., 2-(4-hydroxy-2-ethyl-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 851057], partial [i.e., 2-(4-chloro-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 821008], and high-efficacy [i.e., 2-(6,7-dichloro-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 821010] agonists at a precoupled alpha 2A AR state in the copresence of a G alpha oCys351Ile protein but not G alpha i2Cys352Ile protein by monitoring [35S]GTP gamma S binding responses. Neither positive nor negative efficacy was observed for these compounds by monitoring the adenylate cyclase pathway at a presumably low-affinity alpha 2A AR state. The capacity of the dexefaroxan analogs to antagonize the (-)-epinephrine-mediated [35S]GTP gamma S binding response at a G alpha oCys351Ile protein was inversely correlated with their magnitude of intrinsic activity and unrelated to their ligand binding affinity for the alpha 2A AR. On the other hand, their capacity to antagonize either (-)-epinephrine or 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline tartrate (UK 14304)-mediated inhibition of forskolin-stimulated cAMP formation was not related with the rank order of antagonist capacity for the (-)-epinephrine-mediated [35S]GTP gamma S binding response. In conclusion, these data demonstrate that certain alpha2 AR ligands that are assumed to be antagonists, may yield dissimilar pharmacological responses, dependent on the investigated agonist-stimulated effector pathway.

This study was undertaken to investigate the pharmacology of cloned guinea pig and rat 5-hydroxytryptamine (serotonin; 5-HT)1D receptor sites. Guinea pig, rat, and mouse 5-HT1D receptor genes were cloned, and their amino acid sequences were compared with those of the human, dog, and rabbit. The overall amino acid sequence identity between these 5-HT1D receptors is high and varies between 86 and 99%. The sequence homology is slightly more divergent (13-27%) in the N-terminal extracellular region of these 5-HT1D receptors. Guinea pig and rat 5-HT1D receptors, stably and separately expressed in rat C6 glial cells, are negatively coupled to cyclic AMP formation upon stimulation with agonists, as previously found for cloned human 5-HT1D receptor sites. The cyclic AMP data show some common pharmacological features for the 5-HT1D receptors of guinea pig, rat, and human: an almost similar rank order of potency for the investigated 5-HT1D receptor agonists, stereoselectivity for the binding affinity and agonist potency of R(+)-8-hydroxy-2-(di-n-propylamino)tetralin, and equal 5-HT1D receptor-mediated antagonist potency for methiothepin and the 5-HT2 receptor antagonists ritanserin and ketanserin. In conclusion, the pharmacology of the cloned 5-HT1D receptor subtype seems, unlike the 5-HT1B receptor subtype, conserved among various mammal species such as the human, guinea pig, and rat.

G protein activation mediated by serotonin 5-HT1A and 5-HT(1B/D) receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [35S]GTPgammaS binding to brain sections. [35S]GTPgammaS binding was stimulated by the mixed 5-HT1A/5-HT(1B/D) agonist L694247 in brain structures enriched in 5-HT1A binding sites, i.e., hippocampus (+140 +/- 14%), dorsal raphe (+70 +/- 8%), lateral septum (+52 +/- 12%), cingulate (+36 +/- 8%), and entorhinal cortex (+34 +/- 5%). L694247 caused little or no stimulation of [35S]GTPgammaS binding in brain regions with high densities of 5-HT(1B/D) binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [35S]GTPgammaS binding response was antagonized by WAY100635 (10 microM) and methiothepin (10 microM). In contrast, the 5-HT1B inverse agonist SB224289 (10 microM) did not affect the L694247-mediated [35S]GTPgammaS binding response, and the mixed 5-HT(1B/D) antagonist GR127935 (10 microM) yielded a partial blockade. The distribution pattern of the [35S]GTPgammaS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [35S]GTPgammaS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 microM) stimulated [35S]GTPgammaS binding in the hippocampus by 20-50%. Naratriptan, CP122638, and dihydroergotamine stimulated [35S]GTPgammaS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT1A but not 5-HT(1B/D) receptors can be measured in guinea pig brain sections.

The hypothesis that prolonged treatment of a constitutively active receptor with inverse agonists may lead to increased receptor density was tested for the alpha(2)-adrenoceptor (AR) inverse agonist (+)-RX 811059 at both the wild-type (WT) and Thr(373)Lys alpha(2A) ARs in CHO-K1 cells by monitoring [(3)H]RX 821002 and [(35)S]GTPgammaS binding responses. One-hundred micromolar KCl instead of NaCl in the [(35)S]GTPgammaS membrane binding assay favoured the detection of a high-magnitude constitutive alpha(2A) AR activity. Under this condition, (+)-RX 811059 was an inverse agonist [ E(max) (% vs. basal): Thr(373)Lys alpha(2A) AR (-52+/-2)WT alpha(2A) AR (-31+/-6)] while atipamezole was a silent neutral antagonist for both WT and Thr(373)Lys alpha(2A) ARs. The B(max) value of [(3)H]RX 821002 binding sites to membranes of transfected CHO-K1 cells was90% for the Thr(373)Lys alpha(2A) AR compared with the WT alpha(2A) AR (9.1+/-1.4 pmol/mg protein); K(d) values were similar (1.16+/-0.19 nM and 1.51+/-0.15 nM, respectively). Forty-eight-hours' pre-treatment of cells with either 0.1 microM (+)-RX 811059, 1 microM atipamezole or 1 microM of the efficacious agonist d-medetomidine increased the amount of [(3)H]RX 821002 binding sites of both WT (52%-59%) and mutant (306%-447%) Thr(373)Lys alpha(2A) ARs. The same alpha(2) AR ligands also prevented the loss of [(3)H]RX 821002 binding sites as induced by incubation of transfected CHO-K1 cellular membranes at 37 degrees C for 4 h (WT alpha(2A) AR) and 2 h (Thr(373)Lys alpha(2A) AR); 0.1 microM (+)-RX 811059 and 1 microM atipamezole caused an increase compared with the control amount of [(3)H]RX 821002 binding sites to the Thr(373)Lys alpha(2A) AR by 73% and 50%, respectively. In conclusion, no relationship was found between inverse agonism and alpha(2A) AR up-regulation. It is suggested that this is due to structural stabilisation of the alpha(2A) AR, irrespective of the nature of the ligand.

Real-time analysis of dopamine:antagonist interactions at the recombinant D2short receptor was performed by measuring time-dependent Ca2+ responses following activation of a chimeric Galphaq/o protein in CHO-K1 cells. Terguride (57%), +-UH 232 (20%) and buspirone (16%) demonstrated dopamine-like intrinsic activity at the presumably unoccupied, dopamine-free receptor; remoxipride, pipamperone and L 741626 being silent at 1 microM. Each of the putative antagonists (1 microM) displayed a transient reversal capacity of the low-magnitude Ca2+ phase in the dopamine-bound receptor state ( E(rev): 68%-92% vs. 1 microM tropapride) with a t1/2 between 8.8 s and 13.9 s upon antagonist addition; this capacity was either almost fully [remoxipride, pipamperone and +-UH 232] or partially [buspirone (31%), terguride (45%) and L 741626 (70%)] lost upon further incubation. The biphasic reversal Ca2+ profile of these dopamine antagonists is different from previously characterised dopamine antagonists which display either full reversal of the low-magnitude Ca2+ response with a fast or slow onset of action, or partial reversal stably present over the entire incubation period. The dynamic Ca2+ data strongly suggest that the dopamine D2short receptor can be blocked via multiple molecular mechanisms.

Chimeric Gα proteins, obtained by exchanging their C-terminal portion for that of a Gα protein from an unrelated class, drive the receptor selectivity to that corresponding to the introduced Gα protein domain. The α2A-adrenoceptor (α2AAR), which yielded an efficacious and weak [35S]GTPγS binding response by respectively Gα o and Gα i3 protein, was investigated in CHO-K1 cells co-expressing chimeric Gα proteins for which the six last C-terminal amino acids between Gα o and Gα i3 proteins, and reciprocally, were permuted. Activation of the chimeric Gα o / i3 protein was highly efficient whereas the Gα i3 / o protein yielded a weak stimulation. These [35S]GTPγS binding responses were not different from their parental wild-type Gα o and Gα i3 proteins. Similar results were obtained with an α2AAR carrying a facilitating Thr373Lys mutation in a putative G protein interaction domain. These data indicate that the six terminal Gα o protein amino acids do not constitute a major α2AAR interaction domain for Gα protein activation.

Using a combination of reverse transcription polymerase chain reaction (RT-PCR) and inverse-PCR techniques, we amplified, cloned and sequenced a full-length porcine 5-hydroxytryptamine 1F (5-ht 1F ) receptor complementary DNA (cDNA) derived from porcine trigeminal ganglion. Sequence analysis revealed 1101 base pairs (bp) encoding an open reading frame of 366 amino acids showing a high similarity (>90%) with the 5-ht 1F receptor sequences from other species, including human. The recombinant porcine 5-ht 1F receptor was expressed in African green monkey kidney cell lines (COS-7 cells) and its ligand binding profile was determined using [ 3 H]5-HT. The affinities of several agonists (LY334370 (5-(4-fluorobenzoyl)amino-3-(1-methylpiperidin-4-yl)-1H-indole fumarate)>CP122638 ( N -methyl-3 [pyrrolidin 2( R )-yl methyl]-1H-indol-5-ylmethyl sulphonamide)=naratriptan=5-HT>eletriptan>sumatriptan>frovatriptan=avitriptan>dihydroergotamine>zolmitriptan>5-carboxamidotryptamine>rizatriptan>alniditan=donitriptan>L694247 (2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine) and putative antagonists (methiothepin>GR127935 ( N -[4-methoxy-3-(4-methyl-1-piperazinyl) phenyl]-2′-methyl 4′-(5-methyl-1,2,4-oxadiazol-3-yl) [1,1-biphenyl]-4-carboxamide hydrochloride)>ritanserin>SB224289 (2,3,6,7-tetrahydro-1′-methyl-5-[2′-methyl-4′(5-methyl-1,2,4-oxadiazol-3-yl) biphenyl-4-carbonyl] furo [2,3-f] indole-3-spiro-4′-piperidine hydrochloride)>BRL155572 ([1-(3-chlorophenyl)-4-[3,3-diphenyl (2-( S , R ) hydroxypropanyl)piperazine] hydrochloride)>ketanserin=pindolol) correlated highly with those described for the recombinant human 5-ht 1F receptor (Spearman correlation coefficient; r s =0.942). Nevertheless, as compared to the human homologue, some triptans (i.e. sumatriptan, zolmitriptan and rizatriptan) displayed a 10- to 15-fold lower affinity for the porcine 5-ht 1F receptor. Using RT-PCR technique, the expression of porcine 5-ht 1F receptor mRNA was observed in cerebral cortex, trigeminal ganglion and several blood vessels, but not in skeletal muscles. In conclusion, we have cloned and established the amino acid sequence and ligand binding profile of the porcine 5-ht 1F receptor as well as the distribution of its mRNA. This information may be helpful in exploring the role of 5-ht 1F receptor in physiological processes and diseases, such as migraine.

Background The transferability of economic evaluation in health care is of increasing interest in today’s globalized environment. Here, we propose a methodology for assessing the variability of data elements in cost evaluations in oncology. This method was tested in the context of the European Network of Excellence “Connective Tissues Cancers Network”. Methods Using a database that was previously aimed at exploring sarcoma management practices in Rhône-Alpes (France) and Veneto (Italy), we developed a model to assess the transferability of health cost evaluation across different locations. A nested data structure with 60 final factors of variability (e.g., unit cost of chest radiograph) within 16 variability areas (e.g., unit cost of imaging) within 12 objects (e.g., diagnoses) was produced in Italy and France, separately. Distances between objects were measured by Euclidean distance, Mahalanobis distance, and city-block metric. A hierarchical structure using cluster analysis (CA) was constructed. The objects were also represented by their projections and area of variability through correlation studies using principal component analysis (PCA). Finally, a hierarchical clustering based on principal components was performed. Results CA suggested four clusters of objects: chemotherapy in France; follow-up with relapse in Italy; diagnosis, surgery, radiotherapy, chemotherapy, and follow-up without relapse in Italy; and diagnosis, surgery, and follow-up with or without relapse in France. The variability between clusters was high, suggesting a lower transferability of results. Also, PCA showed a high variability (i.e. lower transferability) for diagnosis between both countries with regard to the quantities and unit costs of biopsies. Conclusion CA and PCA were found to be useful for assessing the variability of cost evaluations across countries. In future studies, regression methods could be applied after these methods to elucidate the determinants of the differences found in these analyses. Electronic supplementary material The online version of this article (doi:10.1186/s12913-014-0537-x) contains supplementary material, which is available to authorized users.

Fusion proteins were constructed between either a wild-type or mutant Thr370Lys alpha2B-adrenoceptor (alpha2B AR) and a mouse Galpha15 protein to analyze ligand-receptor interactions at a receptor/Galpha15 protein density ratio of 1. Activation of the wild-type alpha2B AR-Galpha15 fusion protein in CHO-K1 cells by (-)-adrenaline induced a time- and concentration-dependent (pEC50 = 7.37+/-0.13) increase in the intracellular Ca2+ concentration, which could be antagonized by RX 811059 (pK(B) = 7.55+/-0.15). Whereas d-medetomidine and oxymetazoline were as efficacious agonists as (-)-adrenaline, the following ligands displayed partial agonist properties: BRL 44408atipamezoleclonidineUK 14304BHT 920. A comparison with the mutant Thr370Lys alpha2B AR-Galpha15 fusion protein displayed similar Ca2+ kinetics and a ligand-mediated receptor activation profile characterized by higher potencies and greater maximal Ca2+ responses for the ligands being investigated, including the putative antagonists dexefaroxan and idazoxan. RX 811059 and RX 821002 remained silent. Similar conclusions could be made on enhancement of the ligands' intrinsic activities by coexpression of the mutant Thr370Lys alpha2B AR with either a Galpha15 or Galphao Cys351Ile protein. The Thr370Lys alpha2B AR-Galpha protein interactions may modify the tertiary structure of the mutant receptor in such a way that some putative alpha2 AR antagonists are capable of stabilizing an active receptor conformation, thereby generating positive efficacy.

G-protein activation and Ca 2+ responses by the wild-type D 2short receptor and a mutation Thr 343 Arg, in the distal BBX X B motif of its third intracellular loop, were investigated in CHO-K1 cells in terms of ligand:receptor:G-protein interactions. No evidence was obtained for constitutive, agonist-independent receptor activation, but differences in the ligand-mediated activation profiles of both the wild-type and mutant Thr 343 Arg D 2short receptor were observed. Most of the partial agonists, but not bromocriptine, displayed an enhanced response at the mutant D 2short receptor, suggesting that the mutation brings the receptor in a more active state. This enhancement was apparent both at the level of G-protein activation ([ 35 S]GTPγS binding) and at the effector (Ca 2+ response) and occurred with different G α -proteins. Partial agonists were also found to act differently via the wild-type D 2short receptor depending on the involved G α -protein. Compared with higher efficacy agonists, partial agonists displayed Ca 2+ responses with slower and dissimilar kinetic properties. Lisuride and in particular bromocriptine produced a more potent response in the co-presence of a G αo protein instead of a chimeric G αq/o - or a promiscuous G α15 -protein. S(+)-propylnorapomorphine showed a similar partial response irrespective of the combined G α -protein. Bromerguride and (+)-UH 232 induced weak (16 to 21% versus dopamine) intrinsic activity in the co-presence of a G αq/o -protein in contrast to their silent properties with a G α15 - or a G αo Cys 351 Ile-protein. In conclusion, the present data strongly suggest that multiple activation binding sites are involved with these ligands at the D 2short receptor, and that their activation may be unravelled by either the mutation or co-expressed G α -proteins being investigated.

Antipsychotic drugs may mediate their therapeutic effects not only by preventing the binding of dopamine but also by decreasing the propensity of the dopamine receptor to assume an active R* state. Ligand-mediated activation and blockade of the recombinant human D2long receptor was investigated in CHO-K1 cells upon modulation of its R* state. Both the Ala371Lys (A371K) and Thr372Arg (T372R) D2long receptor mutants could be activated in a ligand-dependent manner via a chimeric Gαq/o protein, and more efficaciously so than with the promiscuous Gα15 protein. Dopamine and partial agonists (Emax: lisuride>>(+)-UH 232≃bromerguride) displayed dissimilar Ca2+ kinetic properties at wild-type and mutant receptors. A371K and T372R D2long receptor mutants demonstrated an attenuated and enhanced maximal response to these partial agonists, respectively. Dopamine antagonists were unable to block the transient high-magnitude Ca2+ phase at the wild-type D2long receptor upon simultaneous exposure to antagonist and dopamine, while full blockade of the low-magnitude Ca2+ phase did occur at a later time (onset-time: haloperidol

Quantitative in situ hybridization on rat coronal brain sections with radiolabelled oligonucleotide probes was performed to investigate the effects of antipsychotic drugs on mRNA levels of regulator of G-protein signalling (RGS) 2 and c-fos. This study demonstrated a similar increase of RGS2 mRNA level in the striatum upon both a single and a 21-day treatment with either haloperidol (2 mg/kg) or risperidone (7.5 mg/kg) in contrast to clozapine (20 mg/kg). Otherwise, the acute c-fos mRNA induction in the striatum was abolished by 74 to 89% upon chronic treatment with either haloperidol or risperidone. In conclusion, the induction of RGS2 mRNA in the striatum, in contrast to the immediate early gene c-fos mRNA, is preserved upon chronic treatment with haloperidol and risperidone.

Using a combination of RT – PCR and inverse-PCR techniques, we amplified, cloned and sequenced a full-length porcine 5-HT1B receptor cDNA derived from porcine cerebral cortex. Sequence analysis revealed 1170 bp encoding an open reading frame of 390 amino acids showing a 95% similarity with the human 5-HT1B receptor. The recombinant porcine 5-HT1B cDNA was expressed in monkey Cos-7 cells and its pharmacological profile was determined by radioligand binding assay using [3H]-GR125743. The affinities of several agonists (L694247>ergotamine 5-carboxamidotryptamine=dihydroergotamine=5-HT>CP122638=zolmitriptan>sumatriptan) and putative antagonists (GR127935>methiothepin>SB224289>>ritanserin>ketanserin BRL15572) correlated highly with those described for the recombinant human 5-HT1B receptor. In membranes obtained from cells co-expressing the porcine 5-HT1B receptor and a mutant GαoCys351Ile protein, 5-HT and zolmitriptan increased, while the 5-HT1B receptor antagonist SB224289 decreased basal [35S]-GTPγS binding, thus showing inverse agonism. The potency of zolmitriptan in the [35S]-GTPγS binding assay (pEC50: 7.64±0.04) agreed with its affinity in displacing the antagonist [3H]-GR125743 (pKi: 7.36±0.07). The 5-HT1B receptor mRNA was observed by RT-PCR in several blood vessels, cerebral cortex, cerebellum and trigeminal ganglion. In situ hybridization performed in frontal cerebral cortex sections revealed the expression of 5-HT1B receptor mRNA in pyramidal cells. In conclusion, we have cloned and established the amino acid sequence, ligand binding profile and location of the porcine 5-HT1B receptor. This information may be useful in exploring the role of 5-HT1B receptor in pathophysiological processes relevant for novel drug discovery in diseases such as migraine. British Journal of Pharmacology (2001) 133, 891–901; doi:10.1038/sj.bjp.0704150

Fourteen different receptor subtypes might be regarded as a diversity that is sufficient to accommodate the wide-ranging physiological roles of 5-hydroxytryptamine (5-HT). However, it is becoming clear that, for 5-HT as for other neurotransmitters, the concept of a receptor as a gatekeeper for a specific cellular process or event is too restrictive. Multiple receptor-mediated biochemical cascades can be activated in cells in response to an agonist by a number of mechanisms. Whereas it is well established that different agonists do not necessarily elicit the same magnitude of response, they probably also select between various possible signal transduction pathways. Receptor signalling may be diverse via a single receptor subtype as a consequence of specific agonist-receptor-G protein interactions. 5-HT receptors are even more heterogeneous when one considers that the amino acid sequence of these receptor subtypes may vary from individual to individual, and that there is an increasing number of receptor isoforms due to alternative splicing and RNA editing of 5-HT receptor transcripts. Activation, in particular constitutive, agonist-independent activation, of some of these receptor isoforms has been reported to be altered. This implies that ligands with similar binding affinities may display different pharmacological properties (partial agonist, antagonist, or inverse agonist) versus these receptor isoforms, depending on their activation state. Therefore, intervention with receptor ligands to modify hampered neurotransmission pathways is a difficult task, and one needs to consider the growing evidence of diversity in G protein-coupled receptor signalling.

Activation of the recombinant human α2C-adrenoceptor (α2C AR) by (−)-adrenaline in CHO-K1 cells transiently co-expressing a chimeric Gαq/i1 protein induced a rapid, transient Ca2+ response with a high-magnitude followed by a low-magnitude phase which continued throughout the recorded time period (15 min). Activation of the α2C AR by various α2 AR agonists revealed the following rank order of high-magnitude Ca2+ response [Emax (%) versus 10 μM (−)-adrenaline]: UK 14304 (102±4)=talipexole (101±3)=(−)-adrenaline (100)=d-medetomidine (98±1)>oxymetazoline (81±4)⋍clonidine (75±5). The methoxy- (RX 821002) and ethoxy-derivatives (RX 811059) of idazoxan and the dexefaroxan analogue atipamezole were fully effective as antagonists of both the high- and the low-magnitude Ca2+ response. However, though acting as full antagonists of the high-magnitude response, the further putative α2 AR antagonists idazoxan (27%), SKF 86466 (29%) and dexefaroxan (59%) reversed the low-magnitude response only partially. In conclusion, kinetic analyses of agonist : antagonist interactions at the α2C AR demonstrate a wide spectrum of partial to complete antagonism of the low-magnitude Ca2+ response for structurally related α2 AR ligands. British Journal of Pharmacology (2000) 131, 1385–1390; doi:10.1038/sj.bjp.0703726

textabstractA cDNA encoding the full-length 5-HT(1D) receptor derived from porcine cerebral cortex was amplified, cloned and sequenced, using guinea-pig 5-HT(1D) receptor coding sequence oligonucleotide primers in reverse transcription-polymerase chain reaction (RT - PCR). The 5' and 3' ends of the porcine 5-HT(1D) receptor cDNA were verified by inverse PCR. Sequence analysis of porcine 5-HT(1D) receptor cDNA revealed an open reading frame of 1134 nucleotides encoding a polypeptide of 377 amino acids having 92% homology with the human 5-HT(1D) receptor and 88 - 90% homology with other species homologues. The porcine 5-HT(1D) receptor cDNA was further subcloned into a mammalian expression vector pcDNA3 and expressed in monkey Cos-7 cells. Radioligand binding assays using either [(3)H]-5-CT or [(3)H]-GR125743 on Cos-7 cell membranes showed that pK(i) values of 14 serotonin ligands were highly correlated with those obtained with the human 5-HT(1D) receptor. Nonetheless, a selective antagonist at the human 5-HT(1D) receptor, BRL15572, only poorly recognized the porcine homologue. Using membranes from cells co-expressing the porcine 5-HT(1D) receptor and rat G(alphail)Cys(351) Ile protein, it was shown that 5-HT and zolmitriptan increased, while ketanserin decreased basal [(35)S]-GTPgammaS binding. The potency of zolmitriptan in the [(35)S]-GTPgammaS binding assay (pEC(50): 8. 46+/-0.08) agreed with its affinity in displacing the radioligands [(3)H]-5-CT and [(3)H]-GR125743 (pK(i): 8.38+/-0.15 and 8.67+/-0.08, respectively). In conclusion, we have established the cDNA sequence and pharmacology of the cloned porcine 5-HT(1D) receptor. This information would be useful in exploring the role of divergent amino acid residues in the receptor-ligand interaction as well as the role of 5-HT(1D) receptor in pathophysiological processes relevant for novel drug discovery in diseases such as migraine.

Molecular cloning and expression of canine (ca) serotonin 5-HT(1B) and ca 5-HT(1D) receptor subtypes showed that besides the lower binding affinity of ketanserin for the ca 5-HT(1D) receptor, the ligand binding profiles were similar to their human homologues. Site-directed mutagenesis studies suggest that a Gln(189) residue in the second extracellular loop of the ca 5-HT(1D) receptor may partially account for the lower binding affinity of ketanserin. The coupling of ca 5-HT(1B) and ca 5-HT(1D) receptor subtypes to the phospholipase C pathway was analyzed by measuring stimulation of inositol phosphate formation in COS-7 cells. Zolmitriptan potently stimulated (EC(50) = 4.9 nM) the inositol phosphate formation at ca 5-HT(1D) receptors in a fully pertussis toxin (PTX)-dependent manner, whereas only a weak PTX-resistant inositol phosphate response (26-29% at 10 microM zolmitriptan) could be detected for the ca 5-HT(1B) receptor at a similar expression level. In contrast, both ca 5-HT(1B) and ca 5-HT(1D) receptor subtypes yielded a similar maximal magnitude of inositol phosphate formation (300-340% at 10 microM zolmitriptan) upon co-expression with a mouse (m) G(alpha15) protein. PTX treatment and co-expression with a beta-adrenergic receptor kinase C-terminal polypeptide partially (20-46%) abolished the m G(alpha15) protein-dependent ca 5-HT(1B) and ca 5-HT(1D) receptor-mediated stimulation of inositol phosphate formation. This study suggests both 5-HT receptor subtypes can activate betagamma subunits of endogenous G(i/o) proteins besides their coupling to recombinant m G(alpha15) protein.

PCR-based overlap extension mutagenesis was applied to introduce a Thr381 to Lys mutation in the α2C-adrenoceptor (α2C AR) coding sequence. This cDNA contains 71% G+C nucleotides and conventional PCR procedures were inefficient in generating a corresponding amplification product. Only the combined use of a pre-PCR denaturation step at 100 °C followed by quick chilling on ice and the addition of 1 M of a commercial GC Melt reagent and 5% (v/v) dimethylsulphoxide with the Advantage GC cDNA PCR kit yielded efficient amplification during the three successive PCR steps of the overlap extension procedure. Transient expression of the mutant Thr381Lys α2C AR in Cos-7 cells was performed to determine the binding profile for a series of α2 AR ligands using [3H]RX 821002.

Constitutive activity of the recombinant human 5-hydroxytryptamine(1B) (5-HT(1B)) receptor (RC code 2.1.5HT.01.B) was investigated by mutagenesis of the BBXXB motif (in which B represents a basic residue and X a non-basic residue) located in the C-terminal portion of the third intracellular loop. In contrast with wild-type 5-HT(1B) receptors, three receptor mutants (Thr(313)-->Lys, Thr(313)-->Arg and Thr(313)-->Gln) increased their agonist-independent guanosine 5'-[gamma-[(35)S]thio]triphosphate binding response by 26-41%. This activity represented approx. 30% of the maximal response induced by 5-HT and could be reversed by the inverse agonists methiothepin and 3-(3-dimethylaminopropyl)-4-hydroxy-N-(4-pyridin-4-yl phenyl)-benzenamide (GR 55562). Enhanced agonist-independent and agonist-dependent 5-HT(1B) receptor activation was provided by co-expression of a pertussis toxin-resistant rat G(o)alpha Cys(351)-->Ile protein. The wild-type 5-HT(1B) receptor displayed a doubling in basal activity, whereas a spectrum of enhanced basal activities (313-571%) was observed with a series of diverse amino acid substitutions (isoleucine, glycine, asparagine, alanine, lysine, phenylalanine, glutamine and arginine) at the 5-HT(1B) receptor position 313 in the presence of pertussis toxin (100 ng/ml). Consequently, the constitutive 5-HT(1B) receptor activity can be modulated by the mutation of Thr(313), and displays a graded range between 11% and 59% of maximal 5-HT(1B) receptor activation by 5-HT. No clear pattern is apparent in the framework of traditionally cited amino acid characteristics (i.e. residue size, charge or hydrophobicity) to explain the observed constitutive activities. The various amino acid substitutions that yielded enhanced activity are unlikely to make similar intramolecular interactions within the 5-HT(1B) receptor. It is hypothesized that the positioning of the junction between the third intracellular loop and transmembrane domain VI is altered by mutation of Thr(313) in the BBXXB motif and thereby unmasks G(alpha)-protein interaction points.

The activity state of G proteins is involved in the ligands' maximal responses that can be produced by activating the 5-HT1A receptor (Pauwels et al., 1997). The present study investigated the ligand responses at the recombinant h 5-HT1A receptor (RC: 2.1.5HT.01A) as mediated by the Galpha(o) protein. Therefore, a fusion protein was constructed between the 5-HT1A receptor and a pertussis toxin resistant rat Galpha(o)Cys351Gly mutant protein to define its pharmacological properties at a receptor: Galpha(o) protein density ratio of 1. Pertussis toxin treatment (100 ng/ml) affected neither the expression of the 5-HT1A receptor fusion protein as measured by [3H] MPPF (3.0+/-0.7 pmol/mg protein) nor the 5-HT-mediated [35S]GTPgammaS binding response (146+/-34 fmol/mg protein) in Cos-7 cells. 8-OH-DPAT (Emax: 55+/-7%) and buspirone (Emax: 22+/-4%) yielded partial agonist activity as compared to 5-HT, whereas WAY 100635 acted as a competitive antagonist (pK(B): 9.75+/-0.17). The magnitude of the 8-OH-DPAT response (Emax, %) was highly dependent on the nature of the amino acid 351 in the C-terminus of the Galpha(o) protein: Ile351 (93+/-4) > Cys351 (79+/-3) > Gly351 (55+/-7). The Emax values (%) of buspirone displayed the following gradient: 69+/-5 approximately/= 62+/-8 > 22+/-4. For comparison, maximal responses of 8-OH-DPAT and buspirone were enhanced versus 5-HT upon co-expression of the 5-HT1A receptor with the respective Galpha(o) proteins, probably due to an altered receptor: Galpha(o) protein density ratio. In conclusion, residue 351 of the rat Galpha(o) protein is involved in determining the magnitude of 5-HT1A receptor activation that ligands can produce at these receptors. Moreover, the fusion protein approach allows quantitative comparisons of the intrinsic activities of ligands between one single receptor subtype with different Galpha protein subtypes.

The present study reports on G-protein activation by recombinant 5-HT receptors and by native 5-HT1A and 5-HT1B receptors in guinea-pig and rat brain using agonist-stimulated [35S]GTPgammaS binding responses mediated by a new 5-HT ligand, a dimer of sumatriptan. Dimerization of sumatriptan increased the binding affinity for h 5-HT1B (pKi: 9.22 vs. 7.79 for sumatriptan), h 5-HT1D (9.07 vs. 8.08) and also h 5-HT1A receptors (7.80 vs. 6.40), while the binding affinity for h 5-ht1E (6.67 vs. 6.19) and h 5-ht1F (7.37 vs. 7.78) receptors was not affected. Sumatriptan dimer (10 microM) stimulated [35S]GTPgammaS binding mainly in the superficial gray layer of the superior colliculi, hippocampus and substantia nigra of guinea-pig and rat coronal brain sections. This fits with the labelling by the 5-HT1B/1D receptor antagonist [3H] GR 125743. The observed [35S]GTPgammaS binding responses in the substantia nigra are likely to be mediated by stimulation of the 5-HT1B receptor subtype, since they were antagonized by the 5-HT1B inverse agonist SB 224289 (10 microM), and not by the 5-HT2A/1D antagonist ketanserin (10 microM). Quantitative assessment of the [35S]GTPgammaS binding responses in the substantia nigra of rat showed highly efficacious responses for both sumatriptan dimer and its monomer. In contrast, less efficacious agonist responses (51+/-10% and 35+/-13%, respectively) were measured in the guinea-pig substantia nigra. This may suggest that the G-protein coupling efficacy of 5-HT1B receptors is different between the substantia nigra of both species. In addition, the sumatriptan dimer also activated guinea-pig and rat hippocampal 5-HT1A receptors with high efficacy in contrast to sumatriptan. Therefore, dimerization of sumatriptan can be considered as a new approach to transform a partial 5-HT1A agonist into a more efficacious agonist. In conclusion, the sumatriptan dimer stimulates G-protein activation via 5-HT1B receptors besides 5-HT1A receptors in guinea-pig and rat brain. The magnitude of the 5-HT1B receptor responses is superior for sumatriptan and its dimer in rat compared to guinea-pig substantia nigra.

1. Replacement of a threonine by a lysine at position 373 in the C-terminal portion of the third intracellular loop of the human alpha2A-adrenergic receptor (alpha2A AR) has been reported to generate a constitutively active mutant receptor in analogy with similar mutations in the alpha1B and beta2 AR (Ren et al., 1993). In the present study, the mutant Thr373Lys alpha2A AR receptor was investigated by measuring the formation of inositol phosphates in either the absence or presence of mouse G(alpha)15 protein in Cos-7 cells. 2. Increased affinity, potency and/or efficacy for the agonists [(-)-adrenaline, UK 14304, clonidine, guanabenz and oxymetazoline] was observed, consistent with a precoupled mutant alpha2A AR: G-protein state. The basal inositol phosphates response was similar at the wild-type (wt) and mutant alpha2A AR, but was enhanced at the mutant alpha2A AR upon co-expression with the mouse G(alpha)15 protein. This enhanced response could not be attenuated in the presence of any of the tested alpha2 AR antagonists (10 microM), suggesting that inverse agonist activity did not occur at the mutant alpha2A AR. 3. Ligands that so far have been identified as antagonists at the wt alpha2A AR demonstrated either no intrinsic activity (MK 912, WB 4101, RS 15385, RX 811059 and RX 821002) or positive efficacy [Emax, % vs. 1 microM UK 14304: dexefaroxan (27+/-7), idazoxan (34+/-9), atipamezole (27+/-4), BRL 44408 (59+/-5) and SKF 86466 (54+/-9)] at the mutant alpha2A AR, but only in the presence of the mouse G(alpha)15 protein. The ligand potencies corresponded with their respective pKi values at the mutant alpha2A AR receptor. 4. The partial agonist effect of SKF 86466 was resistant to pertussis toxin treatment (100 ng ml(-1)) and not affected by co-expression of the rat G(alpha)i1 protein. It was virtually absent in the presence of 10 microM RS 15385. SKF 86466 was without intrinsic activity upon co-expression of the mouse G(alpha)q protein. 5. Some putative alpha2 AR antagonists exerted a partial agonist activity that was highly dependent on the presence of specific G-protein alpha-subunits, suggesting that these ligands cause selective G-protein activation at the mutant alpha2A AR.

The putative coupling between stably expressed recombinant h 5-HT1B or h 5-HT1D receptors and K+ channels which regulate excitability was investigated in C6 glioma cells. Outward K+ currents (IK) were examined in nontransfected C6 glioma cells and in cells expressing cloned h 5-HT1B or h 5-HT1D receptors using the patch-clamp technique in the whole-cell configuration. IK was elicited by a depolarizing step from a holding potential of -60 mV. In C6 glioma cells expressing either recombinant h 5-HT1B or h 5-HT1D receptors, sumatriptan similarly increased IK in a concentration-dependent manner (maximum increase 19.4+/-7.2%, n=8, P

Guanine nucleotide-binding protein-coupled receptors may attain an active conformation in the absence of agonist by spontaneous isomerization and thus yield constitutive, agonist-independent, activity. This has mainly been demonstrated for isolated membranes and recombinant wild-type receptors, and mutant receptors. They generally show remarkable increases in the sensitivity of a biological response. The location of activating mutations both within a single receptor and across receptors is widespread, with changes reported in the seven-transmembrane domains, the second and third intracellular loop. For most of these receptors, examples of ligands defined as inverse agonists have been documented. Regulation of these receptors by inverse agonists opposite to that observed by agonists, and the therapeutic potential of inverse agonists is underlined.

Hybrid molecules built up by covalent coupling of aminopropanol derivatives (especially pindolol) with antidepressant drugs like fluoxetine, paroxetine or milnacipran were found to be potent and silent 5-HT1A antagonists (KB < 1 nM for 7c and 9a).

[35S]GTPgammaS binding responses can be used to measure differences between the intrinsic activity of ligands at human 5-hydroxytrypamine-(1A) (h 5-HT1A) receptors expressed in recombinant cell lines. The maximal [35S]GTPgammaS binding response to 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was lower than that to 5-HT in a recombinant C6-glial membrane preparation and dependent on the GDP concentration: it was attenuated by about 60% vs 5-HT by increasing the concentration of GDP from 0.3 to 30 and 300 microM. Whereas dimerization of 8-OH-DPAT almost did not affect its potency at h 5-HT1A receptors (pEC50: 7.45 and 7.40 for 8-OH-DPAT and its dimer at 30 microM GDP), it increased efficacy at h 5-HT1A receptors. The maximal response to the 8-OH-DPAT dimer was systematically greater than the response to 8-OH-DPAT and identical to that to 5-HT; moreover in contrast to the 8-OH-DPAT monomer, the maximal response to the dimer was unaffected by increasing the GDP concentration. An enhanced 135S]GTPgammaS binding response (44 to 63% vs 8-OH-DPAT) was also observed in the hippocampus, lateral septum, dorsal raphe and cingulate cortex of guinea-pig brain sections using autoradiography of 5-HT1A receptor-activated G-proteins. Hence, the 8-OH-DPAT dimer shows increased efficacy at 5-HT1A receptors compared to 8-OH-DPAT. The differential regulation of the maximal agonist responses by GDP suggests that the [35S]GTPgammaS binding responses to these two ligands could be mediated by different G-protein subtypes upon activation of the 5-HT1A receptor.

New dimers of known 5HT1 ligands (5HT, 1-NP or 8-OH-DPAT) have been prepared and evaluated at human cloned 5HT1 b , 5HT1 d and 5HT1 a receptors. Binding experiments show that all these dimers have better affinities at 5HT 1b 1d receptors than their corresponding monomeric ligands. Studies of inhibition of the forskolin-stimulated c-AMP formation mediated by the human 5HT 1b receptor show that hetero-bivalent ligands [combining an agonist (5HT) with an antagonist (1-NP)] behave as partial agonists while the intrinsic activity of bivalent antagonists (combining two 1-NP residues) was found to be spacer dependent. Surprisingly enough, the dimer of 8-OH-DPAT 6 binds to 5HT 1a , 5HT 1b and 5HT 1d receptors with similar high affinity.

G-protein activation by the 5-ht1F receptor agonist 5-(4-fluorobenzoyl)amino-3-(1-methylpiperidin-4-yl)-1H-indole fumarate (LY334370) was investigated by use of autoradiography of receptor-activated G-proteins in guinea-pig brain sections and [35S]-GTPγS binding responses in cell lines stably expressing human 5-HT1A (h 5-HT1A) receptors. LY334370 (10 μM) caused little or no stimulation of [35S]-GTPγS binding in guinea-pig brain regions enriched in 5-ht1F binding sites (e.g., claustrum, caudate/putamen and thalamic nuclei), as identified by labelling with 10 nM [3H]-sumatriptan plus 10 nM 5-carboxamidotryptamine (5-CT). Application of LY334370 (10 μM) to guinea-pig brain sections resulted in an increase of [35S]-GTPγS binding in hippocampus (123±17%), lateral septum (58±14%), dorsal raphe (57±10%), entorhinal (37±11%) and cingulate cortex (28±10%). This distribution fits with the G-protein activation mediated by 5-HT1A receptors as found with lisuride (10 μM), and labelling of 5-HT1A receptors by 140 pM [125I]-4-(2′-methoxy-phenyl)-1-[2′-(n-2′′-pyridinyl)-p-iodobenzamido]-ethyl-piperazine (p-MPPI). The LY334370-mediated [35S]-GTPγS response was antagonized by the selective, silent 5-HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide (WAY100635, 1 μM) in each of the brain structures investigated. The distribution pattern of the [35S]-GTPγS binding response and the antagonist profile suggest that the LY334370-induced response in guinea-pig brain is mediated by 5-HT1A receptors. The maximal LY334370-induced [35S]-GTPγS binding response (83 to 94%) in membranes of recombinant C6-glial/h 5-HT1A and HeLa/h 5-HT1A cells was close to that of 5-HT, suggesting LY334370 to exert high intrinsic activity at h 5-HT1A receptors. In conclusion, in guinea-pig brain sections and recombinant cell lines the 5-ht1F receptor agonist LY334370 causes G-protein activation that is mediated by 5-HT1A receptors. Caution should be taken when employing this ligand as a putative selective 5-ht1F agonist.

A modified overlap extension technique for the creation of chimeric genes is described: the method consists in three PCR steps. The first step is a conventional PCR reaction, in which oligonucleotide primers are partially complementary at their 5' ends to the adjacent fragments that are fused to create the chimer. The second PCR step consists in the fusion of the PCR fragments generated in the first step using the complementary extremities of the primers. The third step corresponds to the PCR amplification of the fusion product. The final PCR product is a chimeric gene built up with the different amplified PCR fragments. The technique is illustrated by the construction of a chimeric 5- hydroxytryptamine (5-HT, serotonin)1B/D receptor by combining one part of the human 5-HT1B (h5-HT1B) and two parts of the h5-HT1D receptor gene. The chimeric gene expressed in Cos-7 cells yielded similar binding properties as the wild type h5-HT1D receptor. © Rapid Science Ltd. 1998

The guinea-pig recombinant 5-hydroxytryptamine1B (gp 5-HT1B) receptor stably transfected in rat C6-glial cells was characterized by monitoring G-protein activation in a membrane preparation with agonist-stimulated [35S]-GTPγS binding. The intrinsic activity of 5-HT receptor ligands was compared with that determined previously at the human recombinant 5-HT1B (h 5-HT1B) receptor under similar experimental conditions. Membrane preparations of C6-glial/gp 5-HT1B cells exhibited [3H]-5-carboxamidotryptamine (5-CT) and [3H] - N- [4-methoxy-3,4 - methylpiperazin-1-yl) phenyl] -3 - methyl - 4-(4 - pyridinyl)benzamide (GR 125743) binding sites with a pKd of 9.62 to 9.85 and a Bmax between 2.1 to 6.4 fmol mg−1 protein. The binding affinities of a series of 5-HT receptor ligands determined with [3H]-5-CT and [3H]-GR 125743 were similar. Ligand affinities were comparable to and correlated (r2: 0.74, P

A new series of arylpiperazide derivatives of phenylpiperazines of general formula 4 has been prepared and evaluated as 5-HT1B receptor antagonists. In vitro experiments at human cloned 5-HT1B receptors show that these derivatives are potent and selective 5-HT1B receptor antagonists. Among them, compound 4f was found to be orally active, to gain access to the CNS and more importantly to induce an increase in extracellular brain 5-HT upon systemic administration.

A new series of arylpiperazide derivatives of 1-naphthylpiperazine of general formula 4 has been prepared and evaluated as 5-HT1B antagonists. Binding experiments at cloned human 5-HT1A, 5-HT1B, and 5-HT1D receptors show that these derivatives are potent and selective ligands for 5-HT1B/1D subtypes with increased binding selectivity versus the 5-HT1A receptor when compared to 1-naphthylpiperazine (1-NP). Studies of inhibition of the forskolin-stimulated cAMP formation mediated by the human 5-HT1B receptor demonstrate that the nature of the arylpiperazide substituent modulates the intrinsic activity of these 1-NP derivatives. Among them, 2-[[8-(4-methylpiperazin-1-yl)naphthalen-2-yl]oxy] -1-(4-o-tolylpiperazin-1-yl)ethanone (4a) was identified as a potent neutral 5-HT1B antagonist able to antagonize the inhibition of 5-HT release induced by 5-CT (5-carbamoyltryptamine) in guinea pig hypothalamus slices. Moreover, 4a was found to potently antagonize the hypothermia induced by a selective 5-HT1B/1D agonist in vivo in the guinea pig following oral administration (ED50 = 0.13 mg/kg).

1. 5-Hydroxytryptamine-1B (5-HT 1B, formerly designated 5-HT 1D beta) and 5-hydroxy-tryptamine-1D (5-HT 1D, formerly designated 5-HT 1D alpha) receptors are distinct molecular entities that mediate serotonergic neurotransmission. Both are G-protein-coupled receptors without introns in their coding region, negatively coupled to adenylate cyclase; their precise function in human beings remains to be defined. In brain, they are highly enriched in the globus pallidus and the substantia nigra. 2. Presynaptic 5-HT 1B/D receptors take part in the control of the release not only of 5-HT itself, but also of other neurotransmitters-for example, acetylcholine, glutamate, dopamine, noradrenaline and gamma-aminobutyric acid. Selective blockade of central 5-HT 1B/D autoreceptors should facilitate 5-HT neurotransmission and may offer a novel approach to antidepressant therapy. Other 5-HT 1B/D receptors are located postsynaptically; those receptors may be supersensitive in the pathophysiology of obsessive-compulsive disorder and may be a potential target for its treatment. 3. Few if any ligands show selectivity for 5-HT 1B or 5-HT 1D receptors or both. Most pharmacological studies have been performed with nonselective antagonists-for example, metergoline, I-naphthylpiperazine, methiothepin, ketanserin and ritanserin. Recently, a novel series of benzanilides have been reported as the first examples of selective 5-HT 1B/D receptor antagonists. GR 127935, a representative compound of this series, displays mixed agonist-antagonist properties both in vitro and in vivo. It induces upon systemic administration in the guinea pig either an opposite (decrease) effect or a small increase (65%, 5 mg/kg) in the concentration of cortical extracellular 5-HT compared with fluoxetine (218%, 10 mg/kg). The importance of blockade of 5-HT 1B/D receptors in the raphe region, their possible interaction with 5-HT 1A receptors, and consequent inhibition of 5-HT release in terminal 5-HT 1B/D receptor-containing regions are discussed. 4. To find out whether the available so-called 5-HT 1B/D receptor antagonists are indeed antagonists and not partial agonists, efficacy was measured at recombinant human 5-HT 1B and 5-HT 1D receptor sites by using a [35S]-GTP gamma S binding assay to membrane preparations of stably transfected rat C6-glial cell lines. Metergoline and the selective 5-HT 1B/D receptor ligands GR 127935 as well as GR 125743 showed significant intrinsic activity (43% to 69%) at the 5-HT 1D receptor subtype, whereas the nonselective ligand 1-naphthylpiperazine yielded less (15% to 19%) intrinsic activity at both receptor subtypes. In contrast, the nonselective ligands methiothepin, ketanserin and ritanserin are inverse agonists because they displayed negative efficacy (-14% to -28%). Differential blockade of 5-HT 1B/D receptors by neutral antagonists and inverse agonists is discussed in relation to the 5-HT tone on 5-HT 1B/D receptors. 5. It can concluded that 5-HT 1B/D receptor ligands modulate 5-HT neurotransmission through a terminal 5-HT 1B/D receptor. Future work should be directed toward the identification of selective 5-HT 1B and 5-HT 1D receptor ligands that display either neutral antagonist or inverse agonist properties to evaluate the therapeutic potential of 5-HT 1B/D receptor blockade.

The rabbit recombinant saphenous vein 5-hydroxytryptamine1B (rb 5-HT1B) receptor stably transfected in rat C6-glial cells was characterized by measuring adenosine 3′:5′-cyclic monophosphate (cyclic AMP) formation upon exposure to various 5-HT receptor ligands. The effects of agonists and antagonists were compared with their effects determined previously at the human cloned 5-HT1B (h 5-HT1B) receptor under similar experimental conditions. Intact C6-glial cells expressing rb 5-HT1B receptors exhibited [3H]-5-carboxamidotryptamine (5-CT) binding sites with a Kd of 0.80±0.13 nm and a Bmax between 225 to 570 fmol mg−1 protein. The binding affinities of a series of 5-HT receptor ligands determined in a membrane preparation with [3H]-5-CT or [3H]-N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide (GR 125,743) were similar. With the exception of ketanserin, ligand affinities were comparable to those determined at the cloned h 5-HT1B receptor site. rb 5-HT1B receptors were negatively coupled to cyclic AMP formation upon stimulation with 5-HT agonists. Of the several 5-HT agonists tested, 5-CT was the most potent, the potency rank order being: 5-CT>5-HT>zolmitriptan>naratriptan>rizatriptan>sumatriptan>R(+)-8-(hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The maximal responses of these agonists were similar to those induced by 5-HT. The potency of these agonists showed a positive correlation (r2=0.87; P

Summary The design and synthesis of a new series of anilide derivatives of serotonin is described. Binding affinity and intrinsic activity were evaluated at cloned human 5-HTIDα, 5-HTIDβ and 5-HTIA receptors. Modification of the terminal substituent on the aromatic moiety (R1) was investigated and optimal affinity, activity and selectivity for 5-HT1D versus 5-HTIA receptors were obtained for the sulfonamide derivatives 9 and 10. Functional activity was also assessed in the New Zealand white rabbit saphenous vein contraction model, in which most of the compounds behaved as full agonists. Further structural modifications are also described, eg, replacement of the oxygen for carbon atom at the 5-position of the tryptamine moiety or terminal N-dimethylation.

A series of serotonin dimers of formula 4 in which two serotonin moeities are linked together through their 5-hydroxyl residue has been prepared and evaluated as 5-HT(1B/1D) receptor agonists. Binding experiments at cloned human 5-HT(1B), 5-HT(1D), and 5-HT(1A) receptors show that all of these dimers are very potent ligands at 5-HT(1B/1D) receptors with increased binding selectivity vs the 5-HT(1A) receptor when compared to serotonin. Studies of inhibition of the forskolin-stimulated c-AMP formation mediated by the human 5-HT(1B) receptor (formerly the 5-HT(1Dbeta) receptor) demonstrate that all of these serotonin dimers behave as full agonists. Among them, the piperazide derivatives of bis-serotonin, 4g,j, were also identified as very potent agonists in contracting the New Zealand white rabbit saphenous vein (pD2 = 7.6 in each case compared to 5.8 for sumatriptan). Results analysis supports the hypothesis that the important increase in potency of the serotonin dimers can be attributed to the presence of two serotonin pharmacophores in the same molecule, while the enhanced selectivity for 5-HT(1B/1D) receptor subtypes may be due to the position of the spacer attachment to serotonin.

Intrinsic activities of serotonin (5-HT) receptor ligands at cloned human 5-HT 1Dβ receptor sites were determined by measuring cAMP responses in two permanently transfected cell types: rat C6-glial and Chinese hamster ovary (CHO)-K1 cells. Both transfected cell lines expressed a similar 5-HT 1Dβ receptor density (361 to 448 fmol/mg protein) and displayed a number of similar cAMP responses: marked inhibition of forskolinstimulated cAMP formation by 5-HT; a similar agonist potency and efficacy with 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine, bufotenine, sumatriptan, 7-trifluoromethyl-4(4-methyl-1-piperazinyl)-pyrolo-(1,2-a) quinoxaline (CGS 12066B), 5-methoxy-3(1,2,3,6-tetrahydro-4-pyridinyl)1 H -indole (RU 24,969), and tryptamine, their maximal effect being comparable to that of 5-HT; less agonist efficacy with m-trifluoro-phenyl-piperazine; (TFMPP) (it inhibited at most 63% of stimulated cAMP formation); and antagonist activity against the 5-CT-mediated agonist response with methiothepin, 2′-methyl-4-(5-methyl-[1,2,4]oxadiazol-3-yl)-bipheny-l4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide (GR 127,935), and ritanserin. Metergoline and 1-naphtylpiperazine showed different intrinsic activities. In contrast to their pronounced antagonist activity in the transfected CHO-K1 cell line, the antagonist effect was only partial and absent for metergoline and 1-naphtylpiperazine in the transfected C6-glial cell line, respectively. In conclusion, these cell lines are useful as a tool to measure with high sensitivity differences in intrinsic activities of 5-HT receptor ligands and, therefore, discriminate between silent antagonists (no intrinsic activity) and antagonists with intrinsic activity ( i.e. partial agonists), even though this intrinsic activity may be relatively weak.

A series of new arylpiperazide derivatives of serotonin has been prepared and evaluated as 5-HT 1D receptor agonists. Binding experiments at cloned human 5-HT 1Dα , 5-HT 1Dβ , and 5-HT 1A receptors show that all the compounds are very potent and selective ligands for 5-HT 1D receptor subtypes. Functional activity studies (contraction of the New Zealand white rabbit saphenous vein) demonstrate that most of the derivatives behave as full agonists. Among them, the aryl sulfonamide derivative 5q (pD 2 = 8.33 compare to 5.75 for sumatriptan) was also identified as a very potent agonist in inhibiting the forskolin-mediated cyclase coupled to 5-HT 1Dβ receptors (EC 50 = 0.52 nM).

New 5-O-carboxymethyl piperazide derivatives of tryptamine (6a-d) have been prepared in 4 steps from serotonin as 5-HT1D receptors agonists. Binding studies with cloned human 5-HT1Dα and 5-HT1Dβ receptors demonstrate that these derivatives are high affinity ligands at both receptor subtypes and inhibition of forskolin mediated cyclase studies with human 5-HT1Dβ and 5-HT1A receptors show that these compounds are very efficient and selective 5-HT1Dβ agonists.

Radioligand binding measurements were performed in membranes of sheep caudate nucleus using [ 3 H]5-hydroxytryptamine (5-HT). [ 3 H]5-HT labeled a population of high affinity binding sites with a K d of 1.9 ± 0.1 nM and a B max of 19.8 ± 2.2 mol/mg tissue. Combined 5-HT 1D/E binding sites were the predominant 5-HT 1 subtype, accounting for 78% of the total population of 5-HT 1 , binding sites. 5-Carboxamidotryptamine (5-CT) and sumatriptan yielded inhibition curves which best fitted a two-site model with high affinity values of 0.8 and 10.1 nM, and 1000 and 206 nM for their low affinity components. The proportion of the high affinity 5-CT and sumatriptan binding sites was 79 and 72%. The binding affinity profile of 5-HT 1D binding sites [5-CT > 5-HT > d-LSD > 5-MeOT > sumatriptan > RU 24, 969 > metergoline > tryptamine = rauwolscine = methylsergide > yohimbine = methiothepin > TFMPP = 8-OH-DPAT > 2-methyl-5-HT > mCPP = quipazine = CP 93, 129 > ketanserin > (-)-propranolol = haloperidol = ipsapirone]compares well to that reported for 5-HT 1D receptor sites in human caudate and cortex (correlation coefficient: 0.99 and 0.98). The present results indicate that sheep caudate nucleus is a valid tissue for studying interaction of compounds with 5-HT 1D binding sites in the relative absence of 5-HT 1E binding sites.

We investigated the ligand binding properties in vitro of two splice variants of the cloned human dopamine D2 receptor (the 443 and 414 amino acids long forms called D2L and D2S, respectively), expressed in 293 human kidney cells, in comparison with those of the dopamine D2 receptors in rat striatum, nucleus accumbens and tuberculum olfactorium. The new radioligand, [125I]2'-iodospiperone, showed a similar high binding affinity (KD:0.056-0.122 nM) for cloned human D2S and D2L receptors and for the D2 receptors in the three rat brain areas. Binding affinities of 25 dopamine antagonists and of 10 dopamine agonists belonging to different chemical classes were measured. The IC50 values of the antagonists were virtually identical in the five preparations: spiperone was the most potent compound (pIC50 approximately 9.9), remoxipride the least potent one (pIC50 approximately 5.7). The agonists showed similar IC50 values for the cloned human D2S and D2L receptors but their affinity for rat brain D2 receptors was 2- to 5-fold higher. Dopamine showed shallow inhibition curves, the high affinity binding was 10-fold lower for the cloned human D2 receptors than for the rat brain D2 receptors. Addition of stable guanosine-5'-triphosphate (GTP) analogues shifted the D2 receptors in the rat brain tissues to the "low" affinity state, the low affinity binding of dopamine was equal to the affinity for the cloned human receptor. None of the dopamine antagonists or agonists could differentiate between the two splice forms of the cloned human D2 receptors or between the D2 receptors in rat striatal and mesolimbic tissues. The lower apparent affinity of some agonists and of dopamine in the absence of stable GTP analogues suggests a less appropriate receptor G-protein coupling for the cloned human D2 receptors expressed in the 293 human kidney cells. Unexpectedly, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S) reduced the [125I]2'-iodospiperone binding to the D2 receptors by 20-35% in the rat brain tissues and the cloned human D2L receptor, and by 75% to the cloned human D2S receptor. The inhibition in the last case could be prevented partly by submicromolar concentrations of dopamine. The GTP-gamma-S effect is suggested to be due to reduction of disulphide bonds in the receptor. Recent molecular modelling studies indicated an important role of the disulphide bridge between Cys107 at the start of transmembrane domain three and Cys182 in the third extracellular loop, for the binding of dopamine to the D2 receptor.

New 5-O-carboxymethyl piperazide derivatives of tryptamine (6a-d) have been prepared in 4 steps from serotonin as 5-HT1D receptors agonists. Binding studies with cloned human 5-HT1Dα and 5-HT1Dβ receptors demonstrate that these derivatives are high affinity ligands at both receptor subtypes and inhibition of forskolin mediated cyclase studies with human 5-HT1Dβ and 5-HT1A receptors show that these compounds are very efficient and selective 5-HT1Dβ agonists.