Reference Agonist Research Articles

Interaction of D3 preferring agonist (−)-N 6-(2-(4-(biphenyl-4-yl)piperazin-1-yl)ethyl)-N 6-propyl-4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-diamine (D-264) with cloned human D2L, D2S, and D3 receptors: potent stimulation of mitogen-activated protein kinases and G protein-coupled inward rectifier potassium channels

This study aims to determine the effect of the novel D(3) dopamine receptor agonist, D-264, on activation of D(3) and D(2) dopamine receptor signal transduction pathways and cell proliferation. AtT-20 neuroendocrine cells stably expressing human D(2S), D(2L), and D(3) dopamine receptors were treated with D-264 and the coupling of the receptors to mitogen-activated protein kinase (MAPK) and G protein-coupled inward rectifier potassium (GIRK) channels was determined using Western blotting and whole-cell voltage clamp recording, respectively. D-264 potently activated MAPK signaling pathway coupled to D(2S), D(2L), and D(3) dopamine receptors. The activation of MAPK was more pronounced than the reference agonist quinpirole and was longer lasting. D-264 also activated GIRK channels coupled to D(2S), D(2L), and D(3) receptors. In addition, D-264 dose-dependently induced cell proliferation in AtT-D(2L) and AtT-D(3) cells. These results indicate that D-264 robustly activates GIRK channels and MAPK coupled to D(2) and D(3) dopamine receptors in AtT-20 cells. D-264 is also a potent inducer of cell proliferation.

Functional investigation of β-adrenoceptors in human isolated detrusor focusing on the novel selective β3-adrenoceptor agonist KUC-7322

This study aimed to characterize the β-adrenoceptor (β-AR) subtype mediating relaxation of isolated human bladder strips and to explore relaxation by the novel β3-AR-selective agonist KUC-7322 for its relaxant effect on the human isolated detrusor and for its effect on the carbachol (CCh)-induced contractile response. In two parallel studies, relaxation of isolated human bladder strips was tested for the β-AR agonists isoproterenol, clenbuterol, BRL 37344, and KUC-7322. For the isoproterenol and KUC-7322 responses, antagonism by CGP 20712A, ICI 118551, and SR59230A was determined. The potency and efficacy of the reference agonists for detrusor relaxation was in line with their known β3-AR activity. KUC-7322 relative to isoproterenol was a full agonist with a pEC(50) of 5.95 ± 0.09 and 5.92 ± 0.11 in the two studies. SR59230A exhibited antagonism of the expected potency against isoproterenol (apparent pK (B) 7.2) but not against KUC-7322. Neither isoproterenol nor KUC-7322 nor forskolin significantly attenuated CCh-induced contraction. These results suggest that KUC-7322 displays full agonistic activity in relaxing the human detrusor without inhibiting the contraction induced by cholinergic stimulation. These characteristics, if proven in vivo, may be beneficial for the treatment of overactive bladder, as increased bladder capacity with a negligible effect on voiding contractions may be anticipated.

A Novel Nonribose Agonist, LUF5834, Engages Residues That Are Distinct from Those of Adenosine-Like Ligands to Activate the Adenosine A2a Receptor

The recent publication of both the antagonist- and agonist-bound structures of the adenosine A(2A) receptor have revealed much about how a ligand may bind to a receptor and cause the conformational changes associated with agonist-mediated activation. In particular, the agonist-bound structure revealed key interactions between the ribose group of adenosine-derived agonists and amino acids in the receptor binding pocket that lead to receptor activation. However, agonists without a ribose group also exist, and we wondered whether such compounds occupy the same agonist binding site. Therefore we used a mutagenesis approach in this study to investigate the mode of binding of 2-amino-4-(4-hydroxyphenyl)- 6-(1H-imidazol-2-ylmethylsulfanyl)pyridine-3,5-dicarbonitrile (LUF5834), a potent partial agonist without a ribose moiety, compared with the adenosine-derived reference agonist 2-[p-(2-carboxyethyl)phenyl-ethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680). Mutation of the orthosteric residue Phe168 to alanine abrogated the function of both agonists. However, mutation to alanine of residues Thr88 and Ser277 shown by the crystal structures to interact with the ribose group of adenosine-like ligands had no effect on the potency of LUF5834. Furthermore, alanine mutation of Asn253, which makes a hydrogen-bonding interaction with the exocyclic nitrogen of the adenine ring, had minimal effect on LUF5834 affinity but removed agonist activity of this ligand. Mutation of other residues, such as the highly conserved Trp246 or Glu13, had significant deleterious effects on the function of CGS21680 but little effect on LUF5834. In summary, our findings suggest that this class of agonist interacts with distinct residues to activate the receptor compared with classic adenosine derived agonists.

The Antipsychotic Drug Loxapine Is an Opener of the Sodium-Activated Potassium Channel Slack (Slo2.2)

Sodium-activated potassium (K(Na)) channels have been suggested to set the resting potential, to modulate slow after-hyperpolarizations, and to control bursting behavior or spike frequency adaptation (Trends Neurosci 28:422-428, 2005). One of the genes that encodes K(Na) channels is called Slack (Kcnt1, Slo2.2). Studies found that Slack channels were highly expressed in nociceptive dorsal root ganglion neurons and modulated their firing frequency (J Neurosci 30:14165-14172, 2010). Therefore, Slack channel openers are of significant interest as putative analgesic drugs. We screened the library of pharmacologically active compounds with recombinant human Slack channels expressed in Chinese hamster ovary cells, by using rubidium efflux measurements with atomic absorption spectrometry. Riluzole at 500 μM was used as a reference agonist. The antipsychotic drug loxapine and the anthelmintic drug niclosamide were both found to activate Slack channels, which was confirmed by using manual patch-clamp analyses (EC(50) = 4.4 μM and EC(50) = 2.9 μM, respectively). Psychotropic drugs structurally related to loxapine were also evaluated in patch-clamp experiments, but none was found to be as active as loxapine. Loxapine properties were confirmed at the single-channel level with recombinant rat Slack channels. In dorsal root ganglion neurons, loxapine was found to behave as an opener of native K(Na) channels and to increase the rheobase of action potential. This study identifies new K(Na) channel pharmacological tools, which will be useful for further Slack channel investigations.

ChemInform Abstract: Design and Pharmacological Evaluation of PF‐4840154, a Non‐Electrophilic Reference Agonist of the TrpA1 Channel.

AbstractConduction of a high throughput screening leads to the identification of a novel class of non‐covalent TrpA1 agonists that is utilized as a replacement for the well established yet problematic reference agonist Mustard Oil.

Design and pharmacological evaluation of PF-4840154, a non-electrophilic reference agonist of the TrpA1 channel

TrpA1 is an ion channel involved in nociceptive and inflammatory pain. It is implicated in the detection of chemical irritants through covalent binding to a cysteine-rich intracellular region of the protein. While performing an HTS of the Pfizer chemical collection, a class of pyrimidines emerged as a non-reactive, non-covalently binding family of agonists of the rat and human TrpA1 channel. Given the issues identified with the reference agonist Mustard Oil (MO) in screening, a new, non-covalently binding agonist was optimized and proved to be a superior agent to MO for screening purposes. Compound 16a (PF-4840154) is a potent, selective agonist of the rat and human TrpA1 channel and elicited TrpA1-mediated nocifensive behaviour in mouse.

Computational investigation of interactions between human H 2 receptor and its agonists

Type 2 histamine receptor (H 2R) is widely distributed in the body. Its main function is modulating the secretion of gastric acid. Most gastric acid-related diseases are closely associated with it. In this study, a combination of pharmacophore modeling, homology modeling, molecular docking and molecular dynamics methods were performed on human H 2R and its agonists to investigate interaction details between them. At first, a pharmacophore model of H 2R agonists was developed, which was then validated by QSAR and database searching. Afterwards, a model of the H 2R was built utilizing homology modeling method. Then, a reference agonist was docked into the receptor model by induced fit docking. The ‘induced’ model can dramatically improve the recovery ratio from 46.8% to 69.5% among top 10% of the ranked database in the simulated virtual screening. The pharmocophore model and the receptor model matched very well each other, which provided valuable information for future studies. Asp98, Asp186 and Tyr190 played key roles in the binding of H 2R agonists, and direct interactions were observed between the three residues and agonists. Residue Tyr250 could also form a hydrogen bond with H 2R agonists. These findings would be very useful for the discovery of novel and potent H 2R agonists.

Evaluation of Cannabinoid Receptor 2 and Metabotropic Glutamate Receptor 1 Functional Responses Using a Cell Impedance–Based Technology

Recently, new technologies based on biosensors and called label free have been developed. These technologies eliminate the need for using markers and dyes. The authors applied one of these technologies, based on measurement of cell impedance variation, to study the pharmacological profiles of ligands for the cannabinoid receptor 2 (CB2), a Gi-coupled receptor, and for the metabopotropic glutamate receptor 1 (mGluR1), a Gq-coupled receptor. Reference agonists and antagonists/inverse agonists for the 2 receptors were applied to recombinant cell lines and impedance monitored over time. Agonists (JWH133 and CP55940 for CB2; quisqualate, glutamate, 1S-3R-ACPD, and S-3,5-DHPG for mGluR1) triggered a variation of impedance consistent in both potency and efficacy with data obtained using classical assays measuring cAMP or Ca(2+) levels. This effect was not present in the parental nontransfected cell line, confirming specific receptor-mediated response. Application of antagonists (AM630 for CB2; YM298198, SCH1014222, J&J16259685, and CPCCOEt for mGluR1) reduced agonist-induced impedance changes. The only exception was the mGluR1 antagonist BAY367620 that, while active in the Ca(2+) assay, was inactive in the impedance assay. Overall, these results confirm the possibility of using cell impedance-based technology to study the pharmacological profile of ligands acting at G-protein-coupled receptors coupled to different downstream signaling pathways.

Revisiting the complex influences of cannabinoids on motor functions unravels pharmacodynamic differences between cannabinoid agonists

While numerous cannabinoid ligands were historically characterized using the tetrad test (hypomobility, catalepsy, hypothermia, analgesia), only few studies have extensively compared HU 210 and CP 55,940 which are nowadays classically used as reference agonists. Therefore, we herein re-examined the acute and the sustained changes in motor activities mediated by these two agonists in adult rats. As expected for cannabinoid agonists, exposure to either HU 210 or CP 55,940 induced a marked reduction in spontaneous locomotion. This reduction observed as early as 15 min after injection was correlated with the typical rearing and cataleptic responses, and was reversed by co-administration of the CB 1 cannabinoid receptor antagonist SR 141716A. Nevertheless, HU 210, but not CP 55,940, was found to induce persistent responses, lasting for at least 24 h. Also suggesting the involvement of additional targets for HU 210, 10 mg/kg SR 141716A failed to reverse the persistent HU 210-mediated decline in locomotion and rearing, while 1 mg/kg was sufficient to completely abolish the behavioural responses measured 6 h after the injection. Beside pharmacokinetic differences, these data therefore denote distinct pharmacodynamic profiles for HU 210 and CP 55,940. Together, these results suggest that HU 210 displays multicomponent responses that should be taken into account when interpreting data from in vivo/ex vivo studies.

Behavioural evidence of agonist-like effect of isoteoline at 5-HT1B serotonergic receptors in mice.

Isoteoline is a compound of aporphine structure derived from the alkaloid glaucine. Previous studies with isoteoline have shown antagonistic activity at 5-HT(2C) serotonergic receptors. We have investigated whether isoteoline interacts with 5-HT(1B) receptors. An isolation-induced social behavioural deficit test in mice was used as a model of stimulation of these receptors. The deficit in the behaviour of isolated mice in this experimental procedure was reported to be sensitive to 5-HT(1B)-receptor stimulation, since agonists at these receptors are capable of reversing it. In our study, we used N-(3-trifluoromethylphenyl)piperazine (TFMPP) (2 mg kg(-1)) as a reference agonist at these receptor sites. TFMPP completely restored the normal behaviour of the isolated mice. Its effect was prevented by propranolol (4 mg kg(-1)), a beta-adrenergic receptor antagonist with a high affinity for 5-HT(1B) receptors, which was inactive by itself. When isoteoline was given before TFMPP, it did not prevent the effect of the latter. Given alone at doses of 0.25, 1, 4 or 8 mg kg(-1), isoteoline showed an effect of its own to normalize the behaviour of isolated mice. The effect of isoteoline (1 mg kg(-1), i.p.) was antagonized by pretreatment with propranolol, indicating that it was mediated through stimulation of 5-HT(1B) receptors. Repeated treatment with isoteoline (1 mg kg(-1), 2 x 3 days, i.p.) produced tolerance to its effect and significantly attenuated the effect of TFMPP, when animals were tested 16 h after the last injection. In conclusion, the results provided functional evidence of agonist-like activity of isoteoline at the 5-HT(1B) receptors.

A Series of 2,4-Disubstituted Quinolines as a New Class of Allosteric Enhancers of the Adenosine A3 Receptor

The adenosine receptor subfamily consists of the adenosine A(1), A(2A), A(2B), and A(3) receptors, which are localized in a variety of tissues throughout the human body. It is, therefore, a challenge to develop receptor specific ligands with improved tissue selectivity. Allosteric modulators could have these therapeutic advantages over orthosteric ligands. In the present study, a series of 2,4-disubstituted quinolines were synthesized on the basis of the structure of LUF6000 (34). Compound 27 (LUF6096) was able to allosterically enhance agonist binding to a similar extent as 34. In addition, this new compound showed low, if any, orthosteric affinity for any of the adenosine receptors. In a functional assay, compound 27 showed improved activity in comparison to 34, as it increased both the intrinsic efficacy and the potency of the reference agonist Cl-IB-MECA at the human adenosine A(3) receptor.

Signal transduction and functional selectivity of F15599, a preferential post‐synaptic 5‐HT1A receptor agonist

Activation of post-synaptic 5-HT(1A) receptors may provide enhanced therapy against depression. We describe the signal transduction profile of F15599, a novel 5-HT(1A) receptor agonist. F15599 was compared with a chemical congener, F13714, and with (+)8-OH-DPAT in models of signal transduction in vitro and ex vivo. F15599 was highly selective for 5-HT(1A) receptors in binding experiments and in [(35)S]-GTPgammaS autoradiography of rat brain, where F15599 increased labelling in regions expressing 5-HT(1A) receptors. In cell lines expressing h5-HT(1A) receptors, F15599 more potently stimulated extracellular signal-regulated kinase (ERK1/2) phosphorylation, compared with G-protein activation, internalization of h5-HT(1A) receptors or inhibition of cAMP accumulation. F13714, (+)8-OH-DPAT and 5-HT displayed a different rank order of potency for these responses. F15599 stimulated [(35)S]-GTPgammaS binding more potently in frontal cortex than raphe. F15599, unlike 5-HT, more potently and efficaciously stimulated G(alphai) than G(alphao) activation. In rat prefrontal cortex (a region expressing post-synaptic 5-HT(1A) receptors), F15599 potently activated ERK1/2 phosphorylation and strongly induced c-fos mRNA expression. In contrast, in raphe regions (expressing pre-synaptic 5-HT(1A) receptors) F15599 only weakly or did not induce c-fos mRNA expression. Finally, despite its more modest affinity in vitro, F15599 bound to 5-HT(1A) receptors in vivo almost as potently as F13714. F15599 showed a distinctive activation profiles for 5-HT(1A) receptor-mediated signalling pathways, unlike those of reference agonists and consistent with functional selectivity at 5-HT(1A) receptors. In rat, F15599 potently activated signalling in prefrontal cortex, a feature likely to underlie its beneficial effects in models of depression and cognition.

β-Arrestin Recruitment Assay for the Identification of Agonists of the Sphingosine 1-Phosphate Receptor EDG1

beta-Arrestin recruitment assays provide a generic assay platform for drug discovery on G-protein-coupled receptors (GPCRs). The PathHunter assay technology developed by DiscoveRx (Fremont, CA) uses enzyme fragment complementation of beta-galactosidase to measure receptor-beta-arrestin proximity by chemiluminescence. This study describes an agonistic screen on the human endothelial differentiation sphingolipid GPCR 1 (EDG1), also known as S1P1, using PathHunter beta-arrestin recruitment technology. Screening of a collection of 345,052 compounds yielded 2157 agonistic hits. Only 10 of these compounds showed beta-arrestin recruitment activity on a nonrelated receptor, indicating high accuracy and specificity of the assay. The authors show that receptor activation with reference agonists can be detected within the same EDG1 PathHunter cell line at the level of beta-arrestin recruitment, G(i/o) protein-mediated inhibition of cyclic adenosine monophosphate (cAMP), and activation of downstream phosphorylation of extracellular signal-regulated protein kinases. The degree of beta-arrestin recruitment was largely unaffected upon blockade of G(i/o) protein signaling with pertussis toxin, whereas kinetic studies demonstrated a lower rate of beta-arrestin-receptor association. In contrast, inhibition of cAMP and phosphorylation of extracellular signal-regulated protein kinases were fully G(i/o) protein regulated. The data indicate that the beta-arrestin enzyme fragment complementation cell line can be used not only for agonistic screening of GPCRs but also for the identification of "biased ligands" (i.e., compounds that differ in G-protein coupling and beta-arrestin-mediated cellular effects).

Differential modulation of AP‐1‐ and CRE‐driven transcription by cannabinoid agonists emphasizes functional selectivity at the CB1 receptor

Long-term adaptations to pharmacological stimuli frequently originate from modulation of complex intracellular signalling pathways. We previously reported that HU210 and CP55940, two CB1 cannabinoid receptor agonists, induced opposite effects on TH expression. Herein, we characterized their influence on cAMP response element (CRE) and activator protein 1 (AP-1)-mediated regulation of gene transcription. The activity of the agonists was examined on transfected N1E-115 cells in which expression of the luciferase reporter gene was controlled by transcription promoters consisting of repeats of either CRE or AP-1 elements. In addition, the implication of classical signalling pathways was investigated using a variety of kinase inhibitors. Consistent with the CB1-mediated reduction of cAMP accumulation, both ligands decreased CRE-driven luciferase expression with similar potencies. HU210 also exhibited a concentration-dependent reduction of luciferase activity in cells engineered to examine AP-1-controlled transcription, whereas such response was not obtained with CP55940. Responses were all inhibited by SR141716A and were modified in Pertussis toxin-treated cells, suggesting agonist-selective regulations of distinct Gi/o-dependent mechanisms through CB1 receptor activation. Finally, PKC inhibitors efficiently inhibited the paradoxical effect of HU210 on AP-1-mediated transcription, indicating selective regulation of PKC-dependent responses. Together, our results demonstrate that two cannabinoid ligands, commonly used as reference agonists acting on the same receptor with similar affinities, differentially modulate gene transcription through distinct controls of AP-1. This could reflect activation of distinct subsets of Gi/o-proteins, supporting the concept of functional selectivity at CB1 receptors.

Peroxisome proliferator-activated receptor-γ antagonists exhibit potent antiproliferative effects versus many hematopoietic and epithelial cancer cell lines

Peroxisome proliferator-activated receptor-gamma ligands have preclinical and clinical anticancer activity. Most studies in this area address agonists, with relatively few reports on anticancer effects of peroxisome proliferator-activated receptor-gamma antagonists. Thus, we evaluated the two pure peroxisome proliferator-activated receptor-gamma antagonists, T0070907 and GW9662, on a panel of hematopoietic and epithelial cell lines. The peroxisome proliferator-activated receptor-gamma antagonists and a reference agonist (pioglitazone) were tested in an in-vitro proliferation assay on a panel of seven hematopoietic and nine epithelial cancer cell lines, some of which are chemoresistant. Peroxisome proliferator-activated receptor-gamma expression was measured by immunoblotting, as was the effect of treatment with these agents on peroxisome proliferator-activated receptor-gamma levels. The effect of exogenous interleukin-6, an antiapoptotic cytokine, on growth inhibition was evaluated as well as the apoptotic effects of these drugs. The peroxisome proliferator-activated receptor-gamma antagonists showed significantly greater potency on all cell lines (IC50s of 3.2-29.7 versus 26.5-78.7 micromol/l for pioglitazone) and greater maximum growth inhibition. Peroxisome proliferator-activated receptor-gamma levels did not correlate with growth inhibition in this panel of cell lines. Combinations of peroxisome proliferator-activated receptor-gamma antagonists and the agonist actually showed schedule-dependent increases in growth inhibition. Exogenous interleukin-6 did not induce resistance to these agents. Both the antagonists and the agonist induced apoptosis, but only the former drugs showed caspase dependence. These two peroxisome proliferator-activated receptor-gamma antagonists have significantly more potent in-vitro antiproliferative effects versus hematopoietic and epithelial cancer cell lines. This effect does not correlate with peroxisome proliferator-activated receptor-gamma levels, suggesting alternative mechanisms or other targets of action. These findings support further translational studies to explore the mechanism of action and therapeutic potential of this class of agents.

Inverse Agonism and Neutral Antagonism at Wild-Type and Constitutively Active Mutant Delta Opioid Receptors

The delta opioid receptor modulates nociceptive and emotional behaviors. This receptor has been shown to exhibit measurable spontaneous activity. Progress in understanding the biological relevance of this activity has been slow, partly due to limited characterization of compounds with intrinsic negative activity. Here, we have used constitutively active mutant (CAM) delta receptors in two different functional assays, guanosine 5'-O-(3-thio)triphosphate binding and a reporter gene assay, to test potential inverse agonism of 15 delta opioid compounds, originally described as antagonists. These include the classical antagonists naloxone, naltrindole, 7-benzylidene-naltrexone, and naltriben, a new set of naltrindole derivatives, H-Tyr-Tic-Phe-Phe-OH (TIPP) and H-Tyr-TicPsi[CH2N]Cha-Phe-OH [TICP(Psi)], as well as three 2',6'-dimethyltyrosine-1,2,3,4-tetrahydroquinoline-3-carboxylate (Dmt-Tic) peptides. A reference agonist, SNC 80 [(+)-4-[(alphaR)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide], and inverse agonist, ICI 174864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu), were also included. In a screen using wild-type and CAM M262T delta receptors, naltrindole (NTI) and close derivatives were mostly inactive, and TIPP behaved as an agonist, whereas Dmt-Tic-OH and N,N(CH3)2-Dmt-Tic-NH2 showed inverse agonism. The two latter compounds showed negative activity across 27 CAM receptors, suggesting that this activity was independent from the activation mechanism. These two compounds also exhibited nanomolar potencies in dose-response experiments performed on wild-type, M262T, Y308H, and C328R CAM receptors. TICP(Psi) exhibited strong inverse agonism at the Y308H receptor. We conclude that the stable N,N(CH3)2-Dmt-Tic-NH2 compound represents a useful tool to explore the spontaneous activity of delta receptors, and NTI and novel derivatives behave as neutral antagonists.

Design of Cyclopentaisoxazoline Amino Acids as Conformationally Constrained Agonists at Glutamate Receptors

AbstractWe have prepared four isomeric 3‐hydroxycyclopentaisoxazoline amino acids 12−15, which represent analogues of glutamic acid having restricted conformations, through a strategy based on the 1,3‐dipolar cycloaddition of bromonitrile oxide to a suitably protected 1‐aminocyclopent‐2‐enecarboxylic acid. These target compounds proved to be inactive when assayed at ionotropic and metabotropic glutamate receptors, except for 12 which is an agonist primarily at mGluR5 (EC50 = 79 μM), but is less active at mGluR2 and only marginally active at mGluR1. The biological data are accounted for through comparison of the conformational profiles of the test compounds with that of reference agonists, i.e., N‐methyl‐D‐aspartate (NMDA, 2), and 1‐aminocyclopentane‐1,3‐dicarboxylic acids [trans‐ACPD, 10; cis‐ACPD, 11]. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

The OECD program to validate the rat uterotrophic bioassay: an overview.

The Organisation for Economic Co-operation and Development has undertaken an international validation program for the rodent uterotrophic bioassay. This validation program comprised two major parts. The first part was the development of a detailed background review document compiling the existing data on the bioassay's history, the molecular and physiologic basis for the bioassay's mechanistic relevance to detect estrogen agonists and antagonists, a review of important bioassay protocol parameters, and a review of the data generated by in vitro assays, previous uterotrophic bioassays, and developmental and reproductive assays to assess and support the overall predictivity of the uterotrophic bioassay. The second part was an extensive multiyear effort managed by a validation management group to demonstrate the operating characteristics of four protocols. The effort was conducted in two phases. The phase 1 results with the reference agonist ethinyl estradiol (EE) and antagonist ZM 189,154 has been published previously. This Environmental Health Perspectives mini-monograph is devoted to the phase 2 work using five weak estrogen agonists, bisphenol A, genistein, methoxychlor, nonylphenol, and o,p -DDT, as well as the negative substance dibutylphthalate. These data show that all protocols successfully detected increases in uterine weights when a sufficient dose level of the weak agonists was administered, whether the substances were known or provided as coded doses to the laboratory. The data with both the reference EE and all five weak agonists are reproducible over time and under a variety of different experimental conditions (e.g., animal strain, diet, housing, bedding, vehicle, animal age). In conclusion, all protocols now have sufficient data to support their validity.

5-HT1B-receptors and vascular reactivity in human isolated blood vessels: assessment of the potential craniovascular selectivity of sumatriptan.

5-HT1B-receptor mediated vasoconstriction of cranial arteries is a potential mechanism by which 5-HT1B/1D-receptor agonists such as sumatriptan produce their antimigraine effects. 5-HT1B-receptors exist in other blood vessels which may give rise to unwanted vascular effects. Therefore we examined the distribution of 5-HT1B-receptor immunoreactivity (i.r.) in human blood vessels (including target and nontarget vessels) and confirmed the functionality of this receptor protein, by comparing the vasoconstrictor effects of sumatriptan and 5-HT (the endogenous ligand) in isolated vessels. Blood vessels (middle meningeal, pial, temporal and uterine arteries and saphenous veins) were obtained from surgical patients (with consent). Sections of the vessels were prepared for routine immunohistochemical studies using specific 5-HT1B- and 5-HT1D-receptor antibodies. For functional studies, ring segments of the vessels were mounted in organ baths for isometric tension recording. 5-HT1B-receptor i.r. was detected on the smooth muscle layer in middle meningeal, pial and uterine arteries and in saphenous vein and sumatriptan produced contractions in these vessels with potency values (mean pEC50) of 7.00, 7.08, 6.44 and 6.61, respectively, the magnitude of contraction was greatest in the cranial arteries with Emax values of 100.7, 60.3, 23.0 and 35.9%, respectively (expressed as a percentage of the reference agonist 45 mm KCl). 5-HT1B-receptor i.r. was not detected in temporal artery and sumatriptan had no effect in this artery. 5-HT1D-receptor i.r. was not detected in any of the vessels studied. Sumatriptan can evoke vasoconstriction in antimigraine target vessels and also in nontarget vessels through an action at 5-HT1B-rcceptors. Sumatriptan acts preferentially to cause contraction in human cranial arteries compared with the other blood vessels we examined and this effect is likely to be shared by other drugs of this class.

A set-point model with oscillatory behavior predicts the time course of 8-OH-DPAT-induced hypothermia

Agonists for the 5-hydroxytryptamine (HT)(1A) receptor induce a hypothermic response that is believed to occur by lowering of the body's set-point temperature. We have developed a physiological model that can be used to predict the complex time course of the hypothermic response after administration of 5-HT(1A) agonists to rats. In the model, 5-HT(1A) agonists exert their effect by changing heat loss through a control mechanism with a thermostat signal that is proportional to the difference between measured and set-point temperature. Agonists exert their effect in a direct concentration-dependent manner, with saturation occurring at higher concentrations. On the basis of simulations, it is shown that, depending on the concentration and the intrinsic efficacy of a 5-HT(1A) agonist, the model shows oscillatory behavior. The model was successfully applied to characterize the complex hypothermic response profiles after administration of the reference 5-HT(1A) agonists R-8-hydroxy-2-(di-n-propylamino)tetralin (R-8-OH-DPAT) and S-8-OH-DPAT. This analysis revealed that the observed difference in effect vs. time profile for these two reference agonists could be explained by a difference in in vivo intrinsic efficacy.