Block by 5-hydroxytryptamine and apomorphine of recombinant human neuronal nicotinic receptors.

The effects of 5-hydroxytryptamine and apomorphine on human neuronal nicotinic acetylcholine receptor/channels were examined by expressing these channels in Xenopus oocytes. Functional channels were expressed by combining one type of alpha subunits (alpha3 or alpha4) and one type of beta subunits (beta2 or beta4). 5-Hydroxytryptamine (100 microM to 1 mM) and apomorphine (10 to 100 microM) inhibited an inward current activated by acetylcholine in the oocytes expressing the channels. The sensitivity to 5-hydroxytryptamine or apomorphine depended on subunit combinations. When concentration-response relationship was obtained for the acetylcholine-activated current, the maximal response was reduced by these compounds. The inhibition by these compounds exhibited voltage-dependence: the inhibition was augmented at negative potentials. The results suggest that 5-hydroxytryptamine and apomorphine noncompetitively inhibits human recombinant nicotinic acetylcholine receptor/channels, presumably by acting on channel pores.     

The hypocretins are weak agonists at recombinant human orexin-1 and orexin-2 receptors

The pharmacology of the orexin-like peptides, hypocretin-1 and hypocretin-2, was studied in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX(1)) or orexin-2 (OX(2)) receptors by measuring intracellular calcium ([Ca(2+)](i)) using Fluo-3AM. Orexin-A and orexin-B increased [Ca(2+)](i) in CHO-OX(1) (pEC(50)=7. 99+/-0.05 and 7.00+/-0.10 respectively, n=8) and CHO-OX(2) (pEC(50)=8.30+/-0.05 and 8.21+/-0.07 respectively, n=5). However, hypocretin-1 and hypocretin-2 were markedly less potent, with pEC(50) values of 5.31+/-0.04 and 5.41+/-0.04 respectively in CHO-OX(2) cells (n=5). In CHO-OX(1) cells 10 microM hypocretin-1 only elicited a 37.5+/-3.4% response whilst 10 microM hypocretin-2 elicited a 18.0+/-2.1% response (n=8). Desensitisation of OX(1) or OX(2) with orexin-A (100 nM) abolished the response to orexin-A (10 nM) and the hypocretins (10 microM), but not to UTP (3 microM). In conclusion, the hypocretins are only weak agonists at the orexin receptors.     

Phe369(7.38) at human 5-HT7 receptors confers interspecies selectivity to antagonists and partial agonists

Background and purpose:

Human and rat 5-HT₇ receptors were studied with a particular emphasis on the molecular interactions involved in ligand binding, searching for an explanation to the interspecies selectivity observed for a set of compounds. We performed affinity studies, molecular modelling and site-directed mutagenesis, with special focus on residue Phe(7.38) of the human 5-HT₇ receptor [Cys(7.38) in rat].

Experimental approach:

Competition binding studies were performed for seven 5-HT₇ receptor ligands at three different 5-HT₇ receptors. The functional behaviour was evaluated by measuring 5-carboxytryptamine-stimulated cAMP production. Computational simulations were carried out to explore the structural bases in ligand binding observed for these compounds.

Key results:

Competition experiments showed a remarkable selectivity for the human receptor when compared with the rat receptor. These results indicate that mutating Cys to Phe at position 7.38 profoundly affects the binding affinities at the 5-HT₇ receptor. Computational simulations provide a structural interpretation for this key finding. Pharmacological characterization of compounds mr25020, mr25040 and mr25053 revealed a competitive antagonistic behaviour. Compounds mr22423, mr22433, mr23284 and mr25052 behaved as partial agonists.

Conclusions and implications:

We propose that the interspecies difference in binding affinities observed for the compounds at human and rat 5-HT₇ receptors is due to the nature of the residue at position 7.38. Our molecular modelling simulations suggest that Phe(7.38) in the human receptor is integrated in the hydrophobic pocket in the central part of the binding site [Phe(6.51)-Phe(6.52)] and allows a tighter binding of the ligands when compared with the rat receptor.     

Labelling of recombinant human and native rat serotonin 5-HT1A receptors by a novel, selective radioligand, [3H]-S 15535: Definition of its binding profile using agonists, antagonists and inverse agonists

The novel benzodioxopiperazine, 5-HT_1A receptor weak partial agonist, S 15535 (4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine) bound with high affinity and selectivity to membranes of Chinese Hamster Ovary cells stably expressing the human (h) 5-HT_1A receptor (K_i = 0.6 nM versus [³H]-8-hydroxy-dipropylamino-tetralin, [³H]-8-OH-DPAT): its affinity at h5-HT_1A receptors was more than 70-fold higher than its affinity at > 50 other binding sites. S 15535 was tritiated to high specific activity (50 Ci/mmol) and its binding profile characterised. At 22° C, [³H]-S 15535 associated and dissociated from h5-HT_1A receptors with half-times of 2.9 and 5.0 min, respectively, yielding a K_d estimate of 3.6 nM. In saturation binding experiments, [³H]-S 15535 displayed a B_max value for h5-HT_1A receptors (1630 fmol/mg), higher than that obtained with the agonist [³H]-8-OH-DPAT (1023 pmol/mg). Guanylyl imidodiphosphate (GppNHp, 100 μM) reduced the binding of [³H]-S 15535 by only 25% compared with 79% for [³H]-8-OH-DPAT at h5-HT_1A receptors. [³H]-S 15535 also showed high affinity, saturable binding to rat hippocampal membranes (B_max = 820 fmol/mg versus 647 fmol/mg for [³H]-8-OH-DPAT). For both h5-HT_1A and rat 5-HT_1A receptors, the K_i values for competition binding of 15 serotonergic ligands with [³H]-S 15535 was highly correlated with that of [³H]-8-OH-DPAT. However, important differences were also observed. The agonist, 5-hydroxytryptamine (5-HT), displayed biphasic competition curves with [³H]-S 15535 but not with [³H]-8-OH-DPAT at h5-HT_1A receptors. Similarly, the ‘antagonists’, spiperone, methiothepin and (+)butaclamol, showed biphasic competition isotherms versus [³H]-S 15535 but not [³H]-8-OH-DPAT. When [³H]-S 15535 competition binding experiments were carried out in the presence of GppNHp (100 μM) the 5-HT and 8-OH-DPAT competition curves shifted to the right, whereas the spiperone and methiothepin competition curves shifted to the left. In contrast, in the presence of GppNHp, the competition isotherms for N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide (WAY 100,635) were not altered. Taken together, these data show that (i) [³H]-S 15535 is a highly selective 5-HT_1A receptor ligand which labels both G-protein-coupled and uncoupled 5-HT_1A receptors, (ii) antagonists, such as WAY 100,635, which yield monophasic isotherms in competition with both [³H]-agonists and [³H]-antagonists, are not sensitive to the G-protein coupling state of the receptor, but (iii) spiperone and methiothepin behaved as inverse agonists, their competition isotherms with [³H]-S 15535 being modulated in an opposite manner to those of agonists. Received: 12 September 1997 / Accepted: 1 December 1997     

Desensitisation of native and recombinant human urotensin-II receptors

Human urotensin-II (U-II) is an 11-amino-acid cyclic peptide that activates a G_q-coupled receptor named UT. Little is known about the desensitisation profile of this receptor as peptide binding is essentially irreversible. In the present study, we have examined the effects of U-II and carbachol on Ca²⁺ signalling in SJCRH30 rhabdomyosarcoma (receptor density, B_max ~0.1 pmol/mg protein) and human embroynic kidney (HEK)_hUT (B _max ~1.4 pmol/mg protein) cells expressing native and recombinant UT, respectively. In SJCRH30, HEK_hUT and human peripheral blood mononuclear cells induced to express native UT by treatment with 2 μg/ml lipopolysaccharide (LPS), we have measured the effects of U-II treatment on UT mRNA. In SJCRH30 cells, primary stimulation with carbachol (250 μM) did not affect a secondary challenge with U-II (1 μM) and primary challenge with U-II did not affect a secondary challenge with carbachol. In contrast, in HEK_hUT cells, primary stimulation with carbachol (250 μM) reduced a secondary challenge to U-II (1 μM) by 84% and primary challenge with U-II reduced a secondary challenge to carbachol by 76%. Pre-treatment of SJCRH30 cells with U-II reduced UT mRNA after 6 h and this returned to basal after 24 h. In recombinant HEK_hUT cells, UT mRNA expression increased following a 6 h treatment with 1 μM U-II. U-II treatment of naïve un-stimulated peripheral blood mononuclear cells was without effect. However, when UT expression is up-regulated following 15 h of LPS treatment, a 6 h U-II challenge reduced UT mRNA by 66%. In summary, we report differences in the desensitisation profiles of native and recombinant U-II receptors. Design and interpretation of functional experiments are hampered by irreversibility of U-II binding.     

Behavioural profiles of putative 5-hydroxytryptamine receptor agonists and antagonists in developing rats

The effects of a variety of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on behaviour in 5- and 20-day old rat pups have been investigated. Increased locomotion and head-weaving responses were induced in both age groups by 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin; 5-HT1A agonist); 5-MeODMT (5-methoxy-N,N-dimethyltryptamine; 5-HT1) and RU 24969 (5-methoxy-3(1,2,3,6-tetrahydropyrindin-4-yl)-1H-indole; 5-HT1B/5-5HT1A). The putative 5-HT1A-agonist LY165163 (1-2-(4-aminophenyl)ethyl 4-(3-trifluoromethylphenyl)piperazine) also produced hyperactivity in the developing pups. In contrast, locomotion was not affected by buspirone (5-HT1A); mCPP (1-(3-chlorophenyl)piperazine; 5-HT1B/5-HT1C) and DOI (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane; 5-HT2) though buspirone produced a small increase in head-weaving at 5- and 20-days. The full 5-HT syndrome was induced in older animals (but not neonates) by both 8-OH-DPAT and 5-MeODMT. Large doses of buspirone, mCPP and DOI also produced signs of reciprocal forepaw treading and flattened body posture at 20-days. In addition, mCPP induced grooming and stereotyped mouthing, while DOI increased sniffing behaviour in the young rats. Catecholaminergic mechanisms were implicated in the head-weaving and locomotor responses to 8-OH-DPAT and RU 24969, following experiments with a number of monoamine receptor antagonists. Preliminary findings with (-)-pindolol, which was high affinity for 5-HT1-receptors, suggested that this subtype of receptor may play a role in hyperlocomotion induced by RU 24969.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization of glutamatergic agonists and antagonists at recombinant human homomeric and heteromeric kainate receptors in vitro

An increasing body of evidence suggests that native kainate receptors form ion channels from homomeric and heteromeric combinations of five receptor subunits: GluR5, GluR6, GluR7, KA1 and KA2. We have examined the activity of agonists and antagonists at recombinant human kainate receptors expressed in HEK293 cells, using both whole-cell electrophysiological recording and 96-well plate fluo-3 based calcium microfluorimetry (FLIPR). Both homomeric (GluR5 and GluR6) and heteromeric (GluR5/6, GluR5/KA2 and GluR6/KA2) receptors were examined. Heteromeric receptor assemblies showed electrophysiological and pharmacological profiles which were distinct from homomeric channels. Several agonists, including AMPA, ATPA and (S)-5-iodowillardiine, and antagonists, including gamma-D-glutamylaminomethylsulphonic acid (GAMS) and the decahydroisoquinoline compounds LY293558, LY377770 and LY382884, were found to act at GluR5-containing channels while having no effect at GluR6 homomers. AMPA, ATPA and (S)-5-iodowillardiine did activate GluR6/KA2 heteromers, but only as partial agonists. Additionally, ATPA was shown to act as an antagonist at homomeric GluR6 receptors at high concentrations (IC50 approximately 2 mM). Kynurenic acid was also found to differentiate between GluR6 and GluR6/KA2 receptors, antagonizing glutamate at GluR6 (IC50 = 0.4 mM), while having no effect at GluR6/KA2 channels. The results of the current study provide a broad pharmacological characterization of both homomeric and heteromeric recombinant human kainate receptors, and identify which compounds are likely to be useful tools for studying these various receptor subtypes.     

Synthesis and characterization of fluorescent antagonists and agonists for human oxytocin and vasopressin V(1)(a) receptors

The fluoresceinyl (Flu) group has been linked by an amide bond to the side chain amino group at position 8 of (a) two oxytocin (OT) antagonists, to give d(CH(2))(5)[Tyr(Me)(2),Thr(4),Orn(8)(5/6C-Flu),Tyr-NH(2)(9)]VT (Orn(8)(5/6C-Flu)OTA) (1) and desGly-NH(2),d(CH(2))(5)[D- Tyr(2),Thr(4),Orn(8)(5/6C-Flu)]VT (2), and (b) eight Lys(8) and Orn(8) analogues of potent OT agonists, to give d[Lys(8)(5/6C-Flu)]VT (3), d[Thr(4),Lys(8)(5/6C-Flu)]VT (4), [HO(1)][Lys(8)(5/6C-Flu)]VT (5), [HO(1)][Thr(4),Lys(8)(5/6C-Flu)]VT (6), d[Orn(8)(5/6C-Flu)]VT (7), d[Thr(4),Orn(8)(5/6C-Flu)]VT (8), [HO(1)][Orn(8)(5/6C-Flu)]VT (9), and [HO(1)][Thr(4),Orn(8)(5/6C-Flu)]VT (10). The tetramethylrhodamyl (Rhm) group was attached to the precursor peptide of 9 to give [HO(1)][Orn(8)(5/6C-Rhm)]VT (11). All 11 fluorescent peptides were evaluated in human OT and vasopressin V(1a) (vasoconstrictor), V(1b) (pituitary), and V(2) (antidiuretic) receptor binding and functional assays. With K(d) = 6.24, 217, >10000, and >10000 nM for the OT, V(1a), V(1b), and V(2) receptors, peptide 1 is a potent and selective fluorescent OT antagonist and may be useful for specifically labeling OT receptors while peptide 2 exhibits low affinities for all the receptors. The fluorescent peptides 3-10 are all very potent agonists for the human OT receptor. They exhibit the following K(d) values (nM) for the human OT, V(1a), V(1b), and V(2) receptors, respectively: (3) 0.29, 57, 124, >10000; (4) 1.8, 25.5, 150, >10000; (5) 0.34, 13.7, 66, nd (not determined); (6) 0.32, 17.3, 53, >10000; (7) 0.25, 107, 393, >10000; (8) 0.40, 30, 282, >10000; (9) 0.18, 12.2, 126, nd; (10) 0.17, 11.8, 87, >1000; (11) 0.092, 7.36, nd, nd. Peptide 7 exhibits both a high affinity and a high selectivity for human OT receptors. Peptides 7 and 11 were utilized to study the internalization of the OT receptor-ligand complex. Preliminary studies indicate that this process is similar to that observed for the vasopressin V(1a) receptor and differs from that observed for vasopressin V(2) receptors. Some or all of the fluorescent OT antagonists and agonists reported here are very promising new fluorescent ligands for labeling cells which express the human OT receptor and are also useful tools to follow endocytosis of the receptor-ligand complex.     

Fatty alcohol phosphates are subtype-selective agonists and antagonists of lysophosphatidic acid receptors

A more complete understanding of the physiological and pathological role of lysophosphatidic acid (LPA) requires receptor subtype-specific agonists and antagonists. Here, we report the synthesis and pharmacological characterization of fatty alcohol phosphates (FAP) containing saturated hydrocarbon chains from 4 to 22 carbons in length. Selection of FAP as the lead structure was based on computational modeling as a minimal structure that satisfies the two-point pharmacophore developed earlier for the interaction of LPA with its receptors. Decyl and dodecyl FAPs (FAP-10 and FAP-12) were specific agonists of LPA(2) (EC(50) = 3.7 +/- 0.2 microM and 700 +/- 22 nM, respectively), yet selective antagonists of LPA(3) (K(i) = 90 nM for FAP-12) and FAP-12 was a weak antagonist of LPA(1). Neither LPA(1) nor LPA(3) receptors were activated by FAPs; in contrast, LPA(2) was activated by FAPs with carbon chains between 10 and 14. Computational modeling was used to evaluate the interaction between individual FAPs (8 to 18) with LPA(2) by docking each compound in the LPA binding site. FAP-12 displayed the lowest docked energy, consistent with its lower observed EC(50). The inhibitory effect of FAP showed a strong hydrocarbon chain length dependence with C12 being optimum in the Xenopus laevis oocytes and in LPA(3)-expressing RH7777 cells. FAP-12 did not activate or interfere with several other G-protein-coupled receptors, including S1P-induced responses through S1P(1,2,3,5) receptors. These data suggest that FAPs are ligands of LPA receptors and that FAP-10 and FAP-12 are the first receptor subtype-specific agonists for LPA(2).     

Blockade of 5-hydroxytryptamine receptors in the central nervous system by beta-adrenoceptor antagonists.

(±)-propranolol, oxprenolol and pindolol in doses ranging from 0.5 to 5 mg/kg antagonised the head twitch produced in mice by 5HTP. These doses had no effect on the pinna reflex. (+) propranolol was inactive. These β-adrenoceptor antagonists also prevented the induction of sleep in 5-day old chicks by 5HT. Their activities in the two tests were similar to their 5HT receptor blocking potencies in the rat fundus preparation. It is suggested that these β-adrenoceptor antagonists may block some 5HT receptors in the central nervous system.     

Structure and function of GABA(C) receptors: a comparison of native versus recombinant receptors

In less than a decade our knowledge of the GABA(C) receptor, a new type of Cl(-)-permeable ionotropic GABA receptor, has greatly increased based on studies of both native and recombinant receptors. Careful comparison of properties of native and recombinant receptors has provided compelling evidence that GABA receptor rho-subunits are the major molecular components of GABA(C) receptors. Three distinct rho-subunits from various species have been cloned and the pattern of their expression in the retina, as well as in various brain regions, has been established. The pharmacological profile of GABA(C) receptors has been refined and more specific drugs have been developed. Molecular determinants that underlie functional properties of the receptors have been assigned to specific amino acid residues in rho-subunits. This information has helped determine the subunit composition of native receptors, as well as the molecular basis underlying subtle variations among GABA(C) receptors in different species. Finally, GABA(C) receptors play a unique functional role in retinal signal processing via three mechanisms: (1) slow activation; (2) segregation from other inhibitory receptors; and (3) contribution to multi-neuronal pathways.     

Affinity of serotonin receptor antagonists and agonists to recombinant and native alpha1-adrenoceptor subtypes.

Binding affinities of serotonin (5-HT)-receptor antagonists and agonists at human recombinant alpha1-adrenoceptor subtypes (alpha1a-, alpha1b- and alpha1d-subtypes) were examined and compared with the functional affinities obtained in rat caudal artery (alpha1A-subtype), dog carotid artery (alpha1B-subtype) and rat thoracic aorta (alpha1D-subtype). Most of the 5-HT-receptor antagonists and agonists tested showed relatively high affinity to three alpha1-adrenoceptor subtypes. The highest affinity close to 1 nM was seen for NAN-190 (5-HT1A antagonist) in binding and functional studies. 5-Methylurapidil (5-HT1A agonist) and BMY7378 (5-HT1A agonist) showed, respectively, alpha1a(alpha1A)- or alpha1d(alpha1D)-subtype selectivity in both binding and functional affinities, but spiperone (5-HT2A antagonist) showed alpha1b-selectivity only in binding affinity. Functional affinity of ritanserin (5-HT2A antagonist) to the alpha1B-subtype was approximately 500-fold lower than that of affinity to the alpha1b-subtype. The present results show that many 5-HT-receptor antagonists and agonists have high affinity to alpha1-adrenoceptors, but suggest that there is deviation between their functional affinities and binding affinities for some drugs.     

Novel serotonin receptor 2 (5-HT2R) agonists and antagonists: a patent review (2004-2014)

Introduction: Serotonin or 5-hydroxytryptamine (5-HT) is a substance found in plasma, which increases smooth muscle contraction and mediates platelet aggregation. In addition, it is a monoamine neurotransmitter and is implicated in diverse behaviors. The serotonin receptor 2 (5-HT₂) subfamily is best known for biased signaling and is strongly expressed mainly in the brain regions postulated to be involved in the modulation of higher cognitive and affective functions. Modulators of the 5-HT₂ receptor are currently used to treat a variety of diseases including chronic pain and psychonosema. These properties suggest that 5-HT₂ receptors may become an important therapeutic target for the treatment of various pathological conditions.

Areas covered: This review highlights the significant progress that has been made in the discovery and development of 5-HT₂ receptor agonists and antagonists based on an analysis of the patent literature between January 2004 and December 2014.

Expert opinion: Cumulative evidence over the past decade supports the notion that the modulation of 5-HT₂ receptors has a positive effect on human cognition and emotion. Therefore, we suggest that new agonists and antagonists may play an important role in the treatment of disorders such as schizophrenia, addiction and obesity.     

Skimmianine and related furoquinolines function as antagonists of 5-hydroxytryptamine receptors in animals

1 Skimmianine, kokusaginine and confusameline, three furoquinolines extracted from the leaves of Evodia merrillii(Rutaceae), were investigated to characterize their selective effects on subtypes of 5-hydroxytryptamine (5-HT) receptors. 2 In the isolated membranes of rat cerebrocortex, using [³H]-5-HT and [³H]-ketanserin as radioligands, skimmianine and the two other furoquinolines displaced radioligand bindings in a concentration-dependent manner. Lower concentrations were required to affect [³H]-ketanserin binding than [³H]-5-HT binding in the order skimmianine > kokusaginine > confusameline. 3 Furoquinolines inhibited 5-HT-induced contraction mediated by 5-HT₂ receptors in the presence of methiothepin in rat isolated aorta. Also, the combination of furoquinolines with ketanserin showed an additive antagonism. 4 These furoquinolines were inactive on the 5-carboxamidotryptamine-induced relaxation of guinea-pig ileum, a 5-HT₁-mediated event. However, 5-HT-induced contraction via 5-HT₂ receptors was reduced by these furoquinolines in a way similar to that in blood vessels. 5 The failure of these compounds to affect the 5-HT-induced Bezold–Jarisch-like reflex in anaesthetized rats, the major .5-HT₃-mediated action, ruled out an action on 5-HT₃ receptors. 6 The results obtained suggest that three furoquinoline alkaloids may act on 5-HT receptors in animals, more selectively to the 5-HT₂ subtype, in the order of skimmianine > kokusaginine > confusameline.     

Cannabinoid receptor 2 (CB2) agonists and antagonists: a patent update

Introduction: Modulation of the CB₂ receptor is an interesting approach for pain and inflammation, arthritis, addictions, neuroprotection, and cancer, among other possible therapeutic applications, and is devoid of central side effects.

Areas covered: This review highlights the novel scaffolds for CB₂ ligands and the diverse therapeutic applications for CB₂ modulators disclosed in patents published since 2012.

Expert opinion: Structural diversity of CB₂ modulator scaffolds characterized the patent literature. Several CB₂ agonists reached clinical Phase II for pain management and inflammation. Other therapeutic applications need to be explored such as neuroprotection and/or neurodegeneration.     

Functional characterization of agonists at recombinant human 5-HT2A, 5-HT2B and 5-HT2C receptors in CHO-K1 cells.

1. The goal of this study was to characterize the agonist pharmacology of human 5-HT2A, 5-HT2B and 5-HT2C (VSV) receptors expressed in CHO-K1 (Chinese hamster ovary) cells. 2. We used a fluorometric imaging plate reader (FLIPR) which allows rapid detection of rises in intracellular calcium levels upon the addition of agonists. 3. Stimulation of all three receptors by 5-HT caused a robust concentration dependent increase in intracellular calcium levels. No such effect was observed from non-transfected control CHO-K1 cells. 4. The rank order of potency of agonists at the different receptor subtypes varied. Tryptamines, BW-723C86, d-norfenfluramine, Ro 60-0175 and LSD exhibited the following rank order of potency; 5-HT2B>5-HT2C>5-HT2A. Piperazines such as m-Chlorophenylpiperazine (mCPP), ORG-12962, MK-212 and also ORG-37684 exhibited a rank order of potency of 5-HT2C>5-HT2B>5-HT2A. The phenylisopropylamines DOI and DOB had a rank order of 5-HT2A>5-HT2B>5-HT2C. 5. Many agonists tested had partial agonist actions when compared to 5-HT, and a wide range of relative efficacies were exhibited, which was cell line dependent. For example, mCPP had a relative efficacy of 65% at 5-HT2C receptors but <25% at either 5-HT2A or 5-HT2B receptors. 6. Interpretation of literature values of functional assays using different cell lines, different receptor expression levels and different receptor isoforms, is complex. Species differences and the previous use of antagonist radioligands to characterize agonist potency in binding assays emphasizes the importance of studying agonists in the same experiment using the same assay conditions and parental cell lines.     

Cisapride and a structural analogue,R 76186, are 5-hydroxytryptamine4 (5-HT4) receptor agonists on the guinea-pig colon ascendens

Summary The purpose of this study was to investigate whether the effects of cisapride and its close structural analogue R 76186 on the isolated guinea-pig colon ascendens, are mediated through 5-HT₄ receptors.Both cisapride and R 76186 induced contractions in a concentration-dependent fashion, giving monophasic concentration-response curves (cisapride: EC₅₀ = 1.1 × 10^–7 M, maximum effect = 40.3% of methacholine induced (3 × 10^–7 M) contractions; R 76186: EC_5o = 2.4 × 10^–8 M, maximum effect = 52.1%). Blockade of either 5-HT₂ or 5-HT₃ receptors did not affect the responses to cisapride. However, tropisetron (in 5-HT₄ receptor-blocking concentrations), and DAU 6285 and SDZ 205–557, two novel selective 5-HT₄ receptor antagonists, depressed the concentration-response curve to cisapride (to about 50%), and the curve to R 76186 was shifted to the right. The apparent pA₂ values were 6.6 (tropisetron), 6.3 (DAU 6285), and 7.5 (SDZ 205-557). However, none of these antagonisms was purely competitive as higher concentrations of these antagonists depressed the curve to R76186. Desensitization of the 5-HT₄ receptor with 5-methoxytryptamine (5-MeOT) inhibited the responses to cisapride, and abolished those to R 76186. The contractions to cisapride and R76186 were sensitive to mutual antagonism, depressing their concentration-response curves.Conclusions: Both cisapride and R 76186 mediate their contractile effects in the guinea-pig colon ascendens through agonism at the 5-HT₄ receptor, though cisapride also uses a non-5-HT mechanism. R 76 186 is a selective and potent 5-HT₄ receptor agonist. Correspondence to M. R. Briejer at the above address in Belgium