Displacement Of Radioligand Binding Research Articles

Oleamide Analogues as Positive Allosteric Modulators of the Serotonin (5‐HT) 5‑HT 2C and 5‑HT 2A Receptors

The G protein-coupled receptors (GPCRs) control crucial functions in the central nervous system (CNS) and are acknowledged as important medication targets. The serotonin (5-HT) 5-HT2R family (5-HT2AR, 5-HT2BR, 5-HT2CR) mediates a myriad of CNS functions. Of note, 5-HT2CR signaling is implicated in metabolic disorders (e.g., obesity) and a variety of neuropsychiatric disorders (e.g., anxiety, schizophrenia and substance use disorders). Activation of the brain 5-HT2AR mediates psychedelic states and mood and cognitive alterations evoked by hallucinogens, which is attractive for the application of psychedelic compounds in psychiatry. We sought to discover novel modulators to selectively potentiate 5-HT2CR and 5-HT2AR signaling while avoiding the undesired activation of 5-HT2BR, widely known as associated with valvulopathy and pulmonary hypertension adverse effects. The application of positive allosteric modulators (PAMs) is an appealing strategy to modulate 5-HT2CR and 5-HT2AR functional response by binding to a less conserved allosteric site. Our PAM discovery efforts have included a focus on the well-known natural product oleamide, which has been implicated in signaling modulation of 5-HT2AR and 5-HT2CR as well as 5-HT1AR and 5-HT7R, with an overall promiscuous pharmacological profile. Three series of oleamide-derived 5-HT2CR and 5-HT2AR PAMs are designed and synthesized via a fragment-based drug discovery approach. Pharmacological evaluation of novel small molecules was conducted in a cell-based, 5-HT-induced, intracellular calcium (Ca2+) release assay with physiologically relevant 5-HT2CR or 5-HT2AR expression levels. Multiple 5‑HT2CR and 5‑HT2ARPAMs were obtained with a functional increase of 5-HT-induced Ca2+ release by 20% or greater in the in vitro assay. In vitro distribution, metabolism, and pharmacokinetic analyses, off-target radioligand binding displacement studies and physicochemical properties indicate that our 5‑HT2CR and 5‑HT2AR PAMs display promising lead- and drug-like attributes. In silico molecular docking with the 5-HT2CR X-ray crystal structure has identified a potential PAM binding site for the oleamide-derived 5-HT2CR PAMs that is shared with our previously reported PAMs that do not share an oleamide scaffold. In conclusion, we have designed, synthesized, and optimized a novel series of 5‑HT2CR and 5‑HT2AR PAMs with promising in vitropharmacological andphysicochemical profiles as potential neurotherapeutics for impaired serotonergic controlled substance use disorders and other CNS disorders.

Novel Dopamine D4 Receptor‐Selective Compounds Reveal Structure‐Activity Relationships that Engender Agonist Efficacy

The dopamine D4 receptor (D4R) plays important roles in cognition, attention, and decision making. Novel D4R‐selective ligands have promise in medication development for neuropsychiatric conditions, including Alzheimer's disease and substance use disorders. To identify new D4R‐selective ligands, and to understand the molecular determinants of agonist efficacy at D4R, we report a series of eighteen novel ligands based on the classical D4R agonist A‐412997 (2‐(4‐(pyridin‐2‐yl)piperidin‐1‐yl)‐N‐(m‐tolyl)acetamide). We report a series of eighteen novel D4R ligands with varied substitutions on the phenylpiperidinyl ring, linker region, and/or the arylamide moieties. Compounds were profiled using radioligand binding displacement assays, β‐arrestin recruitment assays, cAMP inhibition assays, and molecular dynamics computational modeling. We identified several novel high‐affinity (Ki ≤ 4.3 nM) and D4R‐selective (>100‐fold vs other D2‐like receptors) compounds with diverse partial agonist and antagonist profiles, falling into three structural groups. Group 1 modifications resulted in a gain of efficacy at D2Rs and D3Rs. Group 2 modifications resulted in lower partial agonist efficacy at D4R. Group 3 modifications resulted in high‐potency full antagonists at D4R. These compounds inform receptor‐ligand interactions that control efficacy at D2‐like receptors and may provide insights to targeted drug discovery leading to new compound development and new tools for a better understanding of the role of D4Rs in neuropsychiatric disorders.Support or Funding InformationSupport for this research was provided by High Point University, Fred Wilson School of Pharmacy. Additional support was provided by the National Institute of Neurological Disorders and Stroke‐Intramural Research Program, National Institute on Drug Abuse‐Intramural Research Program (Z1A DA000609), and Rowan University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dopamine D4 Receptor-Selective Compounds Reveal Structure-Activity Relationships that Engender Agonist Efficacy.

The dopamine D4 receptor (D4R) plays important roles in cognition, attention, and decision making. Novel D4R-selective ligands have promise in medication development for neuropsychiatric conditions, including Alzheimer’s disease and substance use disorders. To identify new D4R-selective ligands, and to understand the molecular determinants of agonist efficacy at D4R, we report a series of eighteen novel ligands based on the classical D4R agonist A-412997 (1, 2-(4-(pyridin-2-yl)piperidin-1-yl)-N-(m-tolyl)acetamide). Compounds were profiled using radioligand binding displacement assays, β-arrestin recruitment assays, cyclic AMP inhibition assays, and molecular dynamics computational modeling. We identified several novel D4R-selective (Ki ≤ 4.3 nM and >100-fold vs other D2-like receptors) compounds with diverse partial agonist and antagonist profiles, falling into three structural groups. These compounds highlight receptor–ligand interactions that control efficacy at D2-like receptors and may provide insights into targeted drug discovery, leading to a better understanding of the role of D4Rs in neuropsychiatric disorders.

Conformationally rigid derivatives of WAY-267,464: Synthesis and pharmacology at the human oxytocin and vasopressin-1a receptors

WAY-267,464 (1) and twelve conformationally rigid analogues (3a-f–4a-f) were synthesised, characterised and evaluated in cellular assays with the aim of systematically exploring interactions with the oxytocin receptor (OTR). Each analogue was evaluated in radioligand binding displacement assays at both human OTR and arginine vasopressin 1a receptors (V1aR). Physiological characterisation was determined by whole cell IP1 accumulation assays on stably transfected human embryonic kidney (HEK) cells. Incorporation of the rigid, optionally substituted benzene ring abolished OTR activity and diminished V1aR pharmacology when compared to 1. A general trend was observed in V1aR affinity for the propyl analogues (3d-3f) which identified the ortho-substituted analogue as the best in series (Ki = 251 nM) followed by a decrease in affinity through the meta and para-derivatives (3e; Ki = 874 nM and 3f; Ki = 1756 nM respectively). This study confirms the importance of the central pharmacophoric motifs of WAY-267,464 and illuminates the differences in the binding pocket of the highly conserved OTR and V1aR.

An improved, high-efficiency assay for assessing serum anticholinergic activity using cultured cells stably expressing M1 receptors

Assessments of total anticholinergic activity (SAA) in serum are of considerable interest for its potential involvement in cognitive impairment associated with polydrug states in the elderly and other populations. Such estimations have been based on the displacement of radioligand binding in rat brain tissues. The validity of such measurements has been questioned, as a potentially distorting effect of large serum proteins was identified. We sought to develop a modified assay that would be more efficient and free of this potential confound. Cultured CHO cells stably expressing M1 receptors M1WT3 were used. Binding of 3H-radioligands was conducted in 96-well plates and tested in serum containing known amounts of anticholinergic medications. Effects of endogenous serum proteins were assessed by pre-assay filtration and also by deproteinization with perchloric acid (PCA). Binding of [3H]quinuclidinyl benzilate ([3H]QNB) or [3H]N-methyl-scopolamine ([3H]NMS) to M1WT3 cells proved reliable and equally sensitive to varying concentrations of anticholinergic agents. In agreement with previous findings (Cox, Kwatra, Shetty, & Kwatra, 2009), filtration of proteins heavier than 50kDa essentially reduced SAA values to zero. In contrast, PCA preserved more than 70% of the binding seen untreated cell membranes. Cell-based assays also showed significant signal increases compared to the conventional rat brain-based protocol. Further advantages of the cell-based protocol described here include increased sensitivity and reliability, smaller amounts of radioligand needed, and higher throughput. PCA pretreatment eliminates potential artifacts attributable to serum proteins. This step, together with improvements in efficiency, should contribute significantly to the usefulness of the assay.

Relations between stimulation of mesolimbic dopamine and place conditioning in rats produced by cocaine or drugs that are tolerant to dopamine transporter conformational change.

Dopamine transporter (DAT) conformation plays a role in the effectiveness of cocaine-like and other DAT inhibitors. Cocaine-like stimulants are intolerant to DAT conformation changes having decreased potency in cells transfected with DAT constructs that face the cytosol compared to wild-type DAT. In contrast, analogs of benztropine (BZT) are among compounds that are less affected by DAT conformational change. We compared the displacement of radioligand binding to various mammalian CNS sites, acute stimulation of accumbens shell dopamine levels, and place conditioning in rats among cocaine and four BZT analogs with Cl substitutions on the diphenyl-ether system including two with carboalkoxy substitutions at the 2-position of the tropane ring. Binding assays confirmed high-affinity and selectivity for the DAT with the BZT analogs which also produced significant stimulation of mesolimbic dopamine efflux. Because BZT analogs produced temporal patterns of extracellular dopamine levels different from those by cocaine (3-10 mg/kg, i.p.), the place conditioning produced by BZT analogs and cocaine was compared at doses and times at which both the increase in dopamine levels and rates of increase were similar to those produced by an effective dose of cocaine. Despite this equilibration, none of the BZT analogs tested produced significant place conditioning. The present results extend previous findings suggesting that cocaine-like actions are dependent on a binding equilibrium that favors the outward conformational state of the DAT. In contrast, BZT analogs with reduced dependence on DAT conformation have reduced cocaine-like behavioral effects and may prove useful in development of medications for stimulant abuse.

QSAR Modeling and Data Mining Link Torsades de Pointes Risk to the Interplay of Extent of Metabolism, Active Transport, and hERG Liability

We collected 1173 hERG patch clamp (PC) data (IC50) from the literature to derive twelve classification models for hERG inhibition, covering a large variety of chemical descriptors and classification algorithms. Models were generated using 545 molecules and validated through 258 external molecules tested in PC experiments. We also evaluated the suitability of the best models to predict the activity of 26 proprietary compounds tested in radioligand binding displacement (RBD). Results proved the necessity to use multiple validation sets for a true estimation of model accuracy and demonstrated that using various descriptors and algorithms improves the performance of ligand-based models. Intriguingly, one of the most accurate models uncovered an unexpected link between extent of metabolism and hERG liability. This hypothesis was fairly reinforced by using the Biopharmaceutics Drug Disposition Classification System (BDDCS) that recognized 94% of the hERG inhibitors as extensively metabolized in vivo. Data mining suggested that high Torsades de Pointes (TdP) risk results from an interplay of hERG inhibition, extent of metabolism, active transport, and possibly solubility. Overall, these new findings might improve both the decision making skills of pharmaceutical scientists to mitigate hERG liability during the drug discovery process and the TdP risk assessment during drug development.

CEP-26401 (Irdabisant), a Potent and Selective Histamine H3 Receptor Antagonist/Inverse Agonist with Cognition-Enhancing and Wake-Promoting Activities

CEP-26401 [irdabisant; 6-{4-[3-((R)-2-methyl-pyrrolidin-1-yl)-propoxy]-phenyl}-2H-pyridazin-3-one HCl] is a novel, potent histamine H₃ receptor (H₃R) antagonist/inverse agonist with drug-like properties. High affinity of CEP-26401 for H₃R was demonstrated in radioligand binding displacement assays in rat brain membranes (K(i) = 2.7 ± 0.3 nM) and recombinant rat and human H₃R-expressing systems (K(i) = 7.2 ± 0.4 and 2.0 ± 1.0 nM, respectively). CEP-26401 displayed potent antagonist and inverse agonist activities in [³⁵S]guanosine 5'-O-(γ-thio)triphosphate binding assays. After oral dosing of CEP-26401, occupancy of H₃R was estimated by the inhibition of ex vivo binding in rat cortical slices (OCC₅₀ = 0.1 ± 0.003 mg/kg), and antagonism of the H₃R agonist R-α-methylhistamine- induced drinking response in the rat dipsogenia model was demonstrated in a similar dose range (ED₅₀ = 0.06 mg/kg). CEP-26401 improved performance in the rat social recognition model of short-term memory at doses of 0.01 to 0.1 mg/kg p.o. and was wake-promoting at 3 to 30 mg/kg p.o. In DBA/2NCrl mice, CEP-26401 at 10 and 30 mg/kg i.p. increased prepulse inhibition (PPI), whereas the antipsychotic risperidone was effective at 0.3 and 1 mg/kg i.p. Coadministration of CEP-26401 and risperidone at subefficacious doses (3 and 0.1 mg/kg i.p., respectively) increased PPI. These results demonstrate potent behavioral effects of CEP-26401 in rodent models and suggest that this novel H₃R antagonist may have therapeutic utility in the treatment of cognitive and attentional disorders. CEP-26401 may also have therapeutic utility in treating schizophrenia or as adjunctive therapy to approved antipsychotics.

Stimulation of dopamine release by nicotinic acetylcholine receptor ligands in rat brain slices correlates with the profile of high, but not low, sensitivity α4β2 subunit combination

α4β2 neuronal nicotinic receptors (nAChRs) can exist in high and low sensitivity states possibly due to distinct stoichiometries during subunit assembly: (α4) 2(β2) 3 pentamer (high sensitivity, HS) and (α4) 3(β2) 2 pentamer (low sensitivity, LS). To determine if there is a linkage between HS or LS states and receptor-mediated responses in brain, we profiled several clinically studied α4β2* nAChR agonists for the displacement of radioligand binding to α4β2 [ 3H]-cytisine sites in rat brain membranes, effects on stimulation of [ 3H]-dopamine release from slices of rat prefrontal cortex and striatum, and activation of HS and LS human α4β2 nAChRs expressed in Xenopus laevis oocytes. Binding affinities (p K i) and potency (pEC 50) values for [ 3H]-dopamine release closely correlated with a rank order: varenicline > (−)-nicotine > AZD3480 > dianicline ≅ ABT-089. Further, a good correlation was observed between [ 3H]-dopamine release and HS α4β2 pEC 50 values, but not between [ 3H]-dopamine release and LS α4β2. The relative efficacies of the agonists ranged from full to partial agonists. Varenicline behaved as a partial agonist in stimulating [ 3H]-dopamine release and activating both HS and LS α4β2 nAChRs expressed in oocytes. Conversely, ABT-089, AZD3480 and dianicline exhibited little efficacy at LS α4β2 (<5%), were more effective at HS α4β2 nAChRs, and in stimulating cortical and striatal [ 3H]-dopamine release ≥30%. In the presence of α-conotoxin MII to block α6β2* nAChRs, the α4β2* α-conotoxin-insensitive [ 3H]-dopamine release stimulated by these ligands correlates well with their interactions at HS, but not LS. In summary, this study provides support for HS α4β2* nAChR involvement in neurotransmitter release.

Pharmacological characterization of a novel investigational antimuscarinic drug, fesoterodine, in vitro and in vivo

To investigate the primary pharmacology of fesoterodine (a novel antimuscarinic drug developed for treating overactive bladder) and SPM 7605 (its active metabolite, considered to be the main pharmacologically active principle of fesoterodine in man) against human muscarinic receptor subtypes, and to investigate in vitro and in vivo functional activity of these agents on the rat bladder compared with existing standard agents. The displacement of radioligand binding by fesoterodine, SPM 7605 and standard agents in membrane preparations of Chinese hamster ovary (CHO) cells expressing the different human muscarinic receptors (M1-M5) was characterized. Agonistic and antagonistic activities were studied using different CHO cell lines stably expressing the human recombinant muscarinic receptor subtypes. The effects of fesoterodine and SPM 7605 on isolated bladder strips contracted by carbachol or electrical field stimulation (EFS) were investigated. In vivo the effects of fesoterodine and SPM 7605 on micturition variables were assessed using continuous cystometry in conscious female Sprague-Dawley rats, and compared to those of oxybutynin and atropine. In vitro SPM 7605 potently inhibited radioligand binding at all five human muscarinic receptor subtypes with equal affinity across all five. Fesoterodine had a similar balanced selectivity profile but was less potent than SPM 7605. Both substances were competitive antagonists of cholinergic agonist-stimulated responses in human M1-M5 cell lines and had a similar potency and selectivity profile to the radioligand-binding studies. In rat bladder strips, fesoterodine and SPM 7605 caused a rightward shift of the concentration-response curve for carbachol with no depression of the maximum, and concentration-dependently reduced contractions induced by EFS. The potency of both drugs was similar to that of atropine and oxybutynin. In the presence of the esterase inhibitor neostigmine, the concentration-response curve of fesoterodine was shifted to the right, suggesting that part of the activity was caused by metabolism to SPM 7605 by tissue enzymes. In vivo, low doses (0.01 mg/kg) of fesoterodine and SPM 7605 reduced micturition pressure and increased intercontraction intervals and bladder capacity, but did not affect residual volume. Fesoterodine and its active metabolite, SPM 7605, are nonsubtype selective, competitive antagonists of human muscarinic receptors, but SPM 7605 has greater potency than the parent compound. Pharmacodynamic studies in the rat bladder in vitro confirm the competitive muscarinic antagonist profile of these agents in a native tissue preparation, and in vivo studies in the rat showed effects on bladder function consistent with a muscarinic antagonist profile.

Inhibitory effect of buflomedil on prostate α 1A adrenoceptor in the Wistar rat

The inhibitory effect of buflomedil on α 1A-adrenoceptor (AR) in the prostate of Wistar rat was investigated in this study. Normotensive and spontaneous hypertensive rats were orally fed with buflomedil (150 mg/kg). The drug effects on blood pressure and heart rate were monitored by photoelectric volume oscillometric method. Prostate tissue strips from normotensive rats were contracted in vitro in organ bath by phenylephrine (10 −8 to 10 −2 M). The inhibitory effects of buflomedil (10 −9 to 10 −7 M) on the phenylephrine-induced contractions were measured. Radioligand binding displacement study by buflomedil was performed on rat prostate α 1A-adrenoceptor (AR) and spleen α 1B-AR. Furthermore the effects of buflomedil and WB-4101 on phenylephrine (10 μM) activated uptake of 2-[ 14 C ]-deoxy- d-glucose (2-DG) into C 2C 12 cells were evaluated. The results showed that buflomedil feeding did not alter the systolic blood pressure of either spontaneous hypertensive rats or normal rats. Dose–inhibition curves of phenylephrine-induced prostate contraction demonstrated a higher potency of buflomedil than tamsulosin. Buflomedil displaced [ 3 H ]prazosin binding in a concentration-dependent manner both in rat prostate α 1A-AR and spleen α 1B-AR. The ratio of affinity to α 1A-AR and α 1B-AR for buflomedil was 4.06/6.84, indicating selectivity on α 1A-AR over α 1B-AR. Activation of C 2C 12 cell α 1A-AR by phenylephrine increased the glucose uptake to 116%. Both buflomedil and WB-4101 inhibited the uptake in a concentration-dependent manner. In conclusion, the findings showed that buflomedil has preferential α 1A-AR antagonistic effect to inhibit prostate contraction without significantly affecting the blood pressure.

Molecular Determinants of Tuberoinfundibular Peptide of 39 Residues (TIP39) Selectivity for the Parathyroid Hormone-2 (PTH2) Receptor: N-TERMINAL TRUNCATION OF TIP39 REVERSES PTH2 RECEPTOR/PTH1 RECEPTOR BINDING SELECTIVITY

Molecular Determinants of Tuberoinfundibular Peptide of 39 Residues (TIP39) Selectivity for the Parathyroid Hormone-2 (PTH2) Receptor: N-TERMINAL TRUNCATION OF TIP39 REVERSES PTH2 RECEPTOR/PTH1 RECEPTOR BINDING SELECTIVITY

Dexamethasone potentiates high-affinity beta-agonist binding and g(s)alpha protein expression in airway smooth muscle.

Corticosteroids enhance beta-adrenergic responses by actions at both beta-adrenoceptor (beta-AR) and post-beta-AR sites. The present study investigated the effects of dexamethasone on beta-AR density, high-affinity beta-agonist binding, G(s)alpha and G(i)alpha protein expression, and cAMP responses in bovine tracheal smooth muscle (bTSM). Dexamethasone treatment of cultured bTSM cells increased total beta-AR density 1.6- to 1.9-fold as assessed by the saturation binding of [(3)H]CGP-12177 and by displacement of radioligand binding with isoproterenol. Isoproterenol bound to the beta-AR at two sites, a high-affinity site with a density of 5.9 +/- 1.2 fmol/mg protein and a low-affinity site with a density of 16.9 +/- 1. 0 fmol/mg protein. Dexamethasone increased both high- and low-affinity isoproterenol binding sites to 11.1 +/- 2.2 and 25.9 +/- 2.1 fmol/mg protein, respectively, without influencing agonist binding affinities. Dexamethasone also selectively increased G(s)alpha protein levels from 0.99 +/- 0.14 to 1.46 +/- 0.17 microg/mg protein without affecting G(i)alpha levels. The net effect of these changes was a 1.8-fold increase in maximal isoproterenol-induced cAMP generation in dexamethasone-treated bTSM cells. These findings provide new insights into the corticosteroid regulation of beta-adrenergic signaling pathways in airway smooth muscle.

Antinociceptive and toxic effects of (+)-epibatidine oxalate attributable to nicotinic agonist activity.

1. Epibatidine is an analgesic substance, isolated from the skin of the poisonous frog Epipedobates tricolor, for which the mechanism of action was previously unknown. 2. The IC50 of synthetic (+)-epibatidine oxalate (the naturally occurring isomer) for [3H]-nicotine binding to rat whole-brain membranes was 0.1 nM. The (-)-isomer also exhibited high affinity (IC50 = 0.2 nM). 3. (+)- and (-)-Epibatidine exhibited much lower affinity for displacement of the muscarinic ligand [3H]-N-methylscopolamine binding to rat cortical membranes (Kapp = 6.9 microM and 16.0 microM respectively). The (+)-enantiomer of epibatidine had an antagonist/agonist (NMS/oxo-M) binding ratio of 4.2 This is consistent with a muscarinic antagonist profile. 4. (+)-Epibatidine oxalate (10 microM) did not cause significant (> 30%) displacement of radioligand binding to opioid, excitatory amino acid, benzodiazepine, 5-HT, dopamine, adrenaline or peptide receptors. 5. (+)- and (-)-Epibatidine (5-20 micrograms kg-1 s.c.) doubled response latency in the mouse hot-plate test. Antinociception and behavioural depression induced by (+)-epibatidine (5 micrograms kg-1) was fully blocked by the nicotinic antagonists mecamylamine (2 mg kg-1 s.c.) or dihydro-beta-erythroidine (2 mg kg-1 s.c.). The muscarinic antagonist scopolamine (0.4 and 10 mg kg-1 s.c.) caused partial reversal of antinociception induced by (+)-epibatidine in mice, but not in rats. 6. These findings demonstrate that (+)-epibatidine oxalate salt is a highly selective and potent nicotinic analgesic agent.

β-Adrenergic Receptor Overexpression in the Fetal Rat: Distribution, Receptor Subtypes, and Coupling to Adenylate Cyclase Activity via G-Proteins

Biogenic amines are hypothesized to play a role in the control of cell differentiation. We assessed the development of β-adrenergic receptors and their linkage to adenylate cyclase activity in order to determine whether catecholaminergic cell signaling can take place early in development. On Gestational Day 12, β-receptors were present in rat embryo in concentrations comparable to those in mature adrenergic target tissues; the concentrations climbed fivefold by Gestational Day 18. β-Receptor expression was higher in liver than in heart and brain, as identified both by binding to isolated membrane preparations and by receptor autoradiography; nevertheless, receptor distribution was quite widespread, with labeling visible throughout the fetus. Receptor subtype selectivities (β2 in liver, β1 in heart, predominantly β2 in whole fetus) were already in place in early development, but receptor coupling to adenylate cyclase via G-proteins showed substantial developmental changes. Agonist-induced displacement of radioligand binding showed little or no GTP sensitivity on Gestational Day 12, suggesting relatively poor receptor linkage to G(s). In contrast, by Day 18, GTP produced a large shift in the agonist displacement curve. Receptor stimulation of adenylate cyclase by isoproterenol also showed a developmental spike by Gestational Day 18; the pattern for isoproterenol stimulation was distinct from the ontogeny of adenylate cyclase itself and from stimulation by forskolin-Mn2+ (which bypasses the need for receptors or G-proteins) or by fluoride (which activates G-proteins nonselectively). Thus, β-receptors are highly expressed during fetal development and the receptors are readily capable of modulating intracellular cAMP production. Fetal catecholamines, which are produced and released by the adrenal medulla, extraadrenal chromaffin tissue, and cells that transiently express adrenergic phenotype, can thus have a direct impact on the differentiation of a wide variety of cells.

INTERACTION BETWEEN EPIDERMAL GROWTH FACTOR AND ARGININE VASOPRESSIN IN RENAL MEDULLARY MEMBRANES

1. Epidermal growth factor is a potent mitogen that causes natriuresis, diuresis and inhibition of arginine vasopressin-induced water reabsorption. 2. The aim of this study was to determine any interaction between epidermal growth factor and the V1 (vascular) and/or V2 (antidiuretic) arginine vasopressin receptor subtypes. 3. Radioligand binding displacement assays demonstrated that although arginine vasopressin related peptides displaced both radioligands from renal medullary membranes at low concentrations epidermal growth factor displaced neither. 4. Arginine vasopressin V2 receptor second messenger cyclic adenosine monophosphate (cAMP) production was inhibited by epidermal growth factor (IC50 2 x 10(-7) mol/L) as was sodium fluoride cAMP production but only at much higher concentrations. 5. Therefore the diuretic effect of epidermal growth factor is not via direct antagonism of arginine vasopressin receptors but seems mediated via inhibition of the V2 second messenger system.

Survey on the pharmacodynamics of the new antipsychotic risperidone

This review reports on the pharmacodynamics of the new antipsychotic risperidone. The primary action of risperidone is serotonin 5-HT2 receptor blockade as shown by displacement of radioligand binding (Ki: 0.16 nM), activity on isolated tissues (EC50: 0.5 nM), and antagonism of peripherally (ED50: 0.0011 mg/kg) and centrally (ED50: 0.014 mg/kg) acting 5-HT2 receptor agonists in rats. Risperidone is at least as potent as the specific 5-HT2 receptor antagonist ritanserin in these tests. Risperidone is also a potent dopamine D2 receptor antagonist as indicated by displacement of radioligand binding (Ki: 1.4 nM), activity in isolated striatal slices (IC50: 0.89 nM), and antagonism of peripherally (ED50: 0.0057 mg/kg in dogs) and centrally acting D2 receptor agonists (ED50: 0.056-0.15 mg/kg in rats). Risperidone shows all effects common to D2 antagonists, including enhancement of prolactin release. However, some central effects such as catalepsy and blockade of motor activity occur at high doses only. Risperidone is 4-10 times less potent than haloperidol as a central D2 antagonist in rats and it differs from haloperidol by the following characteristics: predominant 5-HT2 antagonism; LSD antagonism; effects on sleep; smooth dose-response curves for D2 antagonism; synergism of combined 5-HT2/D2 antagonism; pronounced effects on amphetamine-induced oxygen consumption; increased social interaction; and pronounced effects on dopamine (DA) turnover. Risperidone displays similar activity at pre- and postsynaptic D2 receptors and at D2 receptors from various rat brain regions. The binding affinity for D4 and D3 receptors is 5 and 9 times weaker, respectively, than for D2 receptors; interaction with D1 receptors occurs only at very high concentrations. The pharmacological profile of risperidone includes interaction with histamine H1 and alpha-adrenergic receptors but the compound is devoid of significant interaction with cholinergic and a variety of other types of receptors. Risperidone has excellent oral activity, a rapid onset, and a 24-h duration of action. Its major metabolite, 9-hydroxyrisperidone, closely mimics risperidone in pharmacodynamics. Risperidone can be characterized as a potent D2 antagonist with predominant 5HT2 antagonistic activity and optimal pharmacokinetic properties.

Chapter 35: Postsynaptic actions of acetylcholine: the coupling of muscarinic receptor subtypes to neuronal ion channels

In recent years, the application of molecular biological techniques has established that there are at least five subtypes of muscarinic receptor, each with a unique pattern of distribution. Radio-ligand binding displacement studies of antagonist pharmacology of the individual receptors expressed in appropriate host cells have shown that each receptor does have a discrete antagonist profile, but that there are no single antagonists with sufficient selectivity to be clearly diagnostic for any particular receptor type. This has hampered the elucidation of the roles of muscarinic receptor subtypes, particularly, in cells which may simultaneously express more than one subtype, as is probably the case for neurones. The use of cell lines transfected with a gene encoding a single muscarinic receptor subtype has extended the characterization of muscarinic receptor subtypes beyond the pharmacology of the recognition site and has generated an additional classification on the basis of the downstream cellular mechanisms to which each subtype preferentially couples. This approach can clearly provide indications of likely coupling mechanisms of subtypes in, for example, the CNS neurones. Initial biochemical experiments with cell lines, such as the neuroblastoma x glioma hybrid NG108-15 have established two major groupings of muscarinic receptor subtypes, with ml, m3, and m5 receptors coupling preferentially to stimulation of phospholipase C (PLC) through a Pertussis toxin-insensitive G-protein, with subsequent elevation of IP3, while m2 and m4 receptors couple through a Pertussis toxin-sensitive G-protein to preferentially inhibit adenylyl cyclase.This chapter describes the electrophysiological consequences of muscarinic receptor activation using chemically differentiated NG108-15 cells transfected with ml-m4 receptor genes. These cells are especially useful for electrophysiological studies, because they express a range of K + and Ca 2+ channels similar to those found in many neuronal cell types. However, NGl08-15 cells do natively express the m4 muscarinic receptor.

Kappa-opioids decrease excitatory transmission in the dentate gyrus of the guinea pig hippocampus

In the guinea pig hippocampus, kappa 1-opioid binding sites were primarily localized in the molecular layer of the dentate gyrus as shown by autoradiography using either the kappa 1-selective radioligand 3H-U69,593 or the nonselective radioligand 3H-diprenorphine in the presence of unlabeled mu- and delta-blocking ligands. In this region, the electrophysiological effects of kappa 1-receptor activation were identified using extracellular and intracellular recordings of dentate granule cell responses. The amplitude of the extracellularly recorded population spike was reduced by U69,593 with an EC50 of 26 nM; this effect was reversible and blocked by the opioid antagonist naloxone. The kappa 1-selective antagonist norbinaltorphimine also blocked the effect of U69,593 with an apparent equilibrium dissociation constant (Ki) of 0.26 nM determined by Schild analysis in the physiologic assay. This value agreed well with the Ki for norbinaltorphimine at kappa 1-binding sites measured by radioligand binding displacement (0.24 nM). These results indicate that the electrophysiologic response observed was likely mediated by kappa 1-receptors. As seen with U69,593, dynorphin B, an endogenous opioid peptide that is present in the dentate gyrus, also inhibited the population spike response. mu- and delta-selective opioid agonists had no effect on the amplitude of the maximally evoked response. Intracellular recordings of dentate granule cells showed no direct effects of U69,593 on the granule cells themselves. However, analysis of synaptic potentials revealed that U69,593 significantly reduced the amplitude of glutaminergic EPSPs evoked by afferent stimulation without affecting IPSP amplitudes. The specific effect of U69,593 application on granule cell EPSPs indicates that presynaptic kappa 1-receptor activation inhibits glutamate release from perforant path terminals in the molecular layer of the dentate gyrus. These results suggest that endogenous dynorphins present in the granule cells may act as feedback inhibitors of the major excitatory input to the dentate gyrus.

Comparative studies of D-myoinositol 1,4,5-trisphosphate and synthetic 6-deoxy D-myoinositol 1,4,5-trisphosphate: Binding and calcium release activity in rat parotid microsomes

In this study, we report our data on the binding of D-myoinositol(1,4,5)P 3 and of 6-deoxy D-myoinositol(1,4,5)P 3 to a rat parotid microsomal fraction and their effect on Ca 2+ release. The binding affinity and the potency of 6-deoxy Ins(1,4,5)P 3 to induce Ca 2+ release are about 100 times lower than those of Ins(1,4,5)P 3. However, maximal concentrations of both inositol trisphosphates induce similar calcium efflux and present comparable displacement of radioligand binding. Experiments were performed to exclude that the microsomal preparations used display rapid metabolism of Ins(1,4,5)P 3 or 6-deoxy Ins(1,4,5)P 3 during binding and Ca 2+ release. We also report that, in permeabilized rat parotid acini preparations, 6-deoxy Ins(1,4,5)P 3 is about 100 times less potent that Ins(1,4,5)P 3 in inducing Ca 2+ release. These data indicate that removal of the hydroxyl group in position 6 of the Ins(1,4,5)P 3 molecule severely reduces its binding affinity which seems, in a large part at least, responsible for the reported loss of potency in mobilizing Ca 2+. Nevertheless, 6-deoxy Ins(1,4,5)P 3 seems to be a full agonist for the release of Ca 2+.