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Opioid-induced constipation (OIC), a typical side effect of opioids, is due to activation of the μ-opioid receptors in the enteric nervous system. Peripherally acting μ-opioid receptor antagonists (PAMORAs) can reverse OIC by inhibiting the peripheral action of opioids without affecting centrally mediated analgesia. Naldemedine is a PAMORA with potent antagonist activity against μ-, δ-, and κ-opioid receptors. In this study, the pharmacological profiles of naldemedine, compared with those of naloxone and naloxegol, were evaluated. In vitro, Schild plot analysis indicated that naldemedine was a noncompetitive antagonist of μ-opioid receptors, whereas other compounds were competitive antagonists. Also, naldemedine showed slower association and dissociation kinetics than the other compounds. In vivo, naldemedine dose-dependently ameliorated morphine-induced inhibition of small intestinal transit (SIT). The dose-response curve was not shifted at 1 and 3 mg/kg morphine. On the contrary, that of naloxegol was significantly shifted to the right from 1 to 3 mg/kg morphine. In morphine-dependent rats, naldemedine caused peripheral withdrawal symptoms (diarrhea) at doses higher than 1 mg/kg, whereas the dose that produced half the maximal preventive effect (ED50) against constipation was 0.03 mg/kg. Naldemedine showed slower onset and a lesser severity of diarrhea than the other compounds at close to the ED50 value in the SIT model. Our results reveal that naldemedine has different pharmacological profiles (type of antagonism and binding kinetics) to the other compounds. This might explain the differential inhibition of morphine-induced SIT and withdrawal symptoms among the three antagonist compounds. SIGNIFICANCE STATEMENT: Naldemedine is a novel peripherally acting μ-opioid receptor antagonist with potent antagonist activity against μ-, δ-, and κ-opioid receptors. Naldemedine showed a noncompetitive antagonism and slower association and dissociation kinetics against μ-opioid receptors than naloxone and naloxegol. Naldemedine showed insurmountable antagonism of morphine-induced inhibition and lower and slower peripheral withdrawal symptoms (diarrhea) than the other compounds. Therefore, naldemedine has a different pharmacological profile (the type of antagonism and binding kinetics) to the other compounds.

GIP(3-30)NH2 is a high affinity antagonist of the GIP receptor (GIPR) in humans inhibiting insulin secretion via G protein-dependent pathways. However, its ability to inhibit G protein-independent signaling is unknown. Here we determine its action on arrestin-recruitment and receptor internalization in recombinant cells. As GIP is adipogenic, we evaluate the inhibitory actions of GIP(3-30)NH2 in human adipocytes. Finally, we determine the receptor selectivity of GIP(3-30)NH2 among other human and animal GPCRs. cAMP accumulation and β-arrestin 1 and 2 recruitment were studied in transiently transfected HEK293 cells and real-time internalization in transiently transfected HEK293A and in HEK293A β-arrestin 1 and 2 knockout cells. Furthermore, human subcutaneous adipocytes were assessed for cAMP accumulation following ligand stimulation. Competition binding was examined in transiently transfected COS-7 cells using human 125I-GIP(3-30)NH2. The selectivity of human GIP(3-30)NH2 was examined by testing for agonistic and antagonistic properties on 62 human GPCRs. Human GIP(3-30)NH2 inhibited GIP(1-42)-induced cAMP and β-arrestin 1 and 2 recruitment on the human GIPR and Schild plot analysis showed competitive antagonism with a pA2 and Hill slope of 16.8 nM and 1.11 ± 0.02 in cAMP, 10.6 nM and 1.15 ± 0.05 in β-arrestin 1 recruitment, and 10.2 nM and 1.06 ± 0.05 in β-arrestin 2 recruitment. Efficient internalization of the GIPR was dependent on the presence of either β-arrestin 1 or 2. Moreover, GIP(3-30)NH2 inhibited GIP(1-42)-induced internalization in a concentration-dependent manner and notably also inhibited GIP-mediated signaling in human subcutaneous adipocytes. Finally, the antagonist was established as GIPR selective among 62 human GPCRs being species-specific with high affinity binding to the human and non-human primate (Macaca fascicularis) GIPRs, and low affinity binding to the rat and mouse GIPRs (Kd values of 2.0, 2.5, 31.6 and 100 nM, respectively). In conclusion, human GIP(3-30)NH2 is a selective and species-specific GIPR antagonist with broad inhibition of signaling and internalization in transfected cells as well as in human adipocytes.

Aim The neuropeptide calcitonin gene-related peptide (CGRP) is found in afferent sensory nerve fibers innervating the resistance arteries and plays a pivotal role in a number of neurovascular diseases such as migraine and subarachnoid bleedings. The present study investigates the binding and antagonistic characteristics of small non-peptide CGRP receptor antagonists (i.e. gepants) in isolated rat brain and mesenteric resistance arteries. Methods The antagonistic behavior of gepants was investigated in isolated rat mesenteric arteries using a wire myograph setup while binding of gepants to CGRP receptors was investigated in rat brain membranes using a radioligand competitive binding assay. Furthermore, the histological location of the key components of CGRP receptor (RAMP1 and CLR) was assessed by immunohistochemistry. Results Our functional studies clearly show that all gepants are reversible competitive antagonists producing Schild plot slopes not significantly different from unity and thus suggesting presence of a uniform CGRP receptor population in the arteries. A uniform receptor population was also confirmed by radioligand competitive binding studies showing similar affinities for the gepants in rat brain and mesenteric arteries, the exception being rimegepant which had 50-fold lower affinity in brain than mesenteric arteries. CLR and RAMP1 were shown to be located in both vascular smooth muscle and endothelial cells of rat mesenteric arteries by immunohistochemistry. Conclusion The present results indicate that, despite species differences in the CGRP receptor affinity, the antagonistic nature of these gepants, the distribution pattern of CGRP receptor components and the mechanism behind CGRP-induced vasodilation seem to be similar in resistance-sized arteries of human and rats.

Positive allosteric modulation of the GABAB receptor is a promising alternative to direct activation of the receptor as a therapeutic approach for treatment of addiction, chronic pain, anxiety, epilepsy, autism, Fragile X syndrome, and psychosis. Here we describe in vitro and in vivo characterization of a novel, potent and selective GABAB positive allosteric modulator (PAM) N-(5-(4-(4-chloro-3-fluorobenzyl)-6-methoxy-3,5-dioxo-4,5-dihydro-1,2,4-triazin-2(3H)-yl)-2-fluorophenyl)acetamide (ADX71441). In vitro, Schild plot and reversibility tests at the target confirmed PAM properties of the compound. In mice and rats ADX71441 is bioavailable after oral administration and is brain penetrant. A single dose of ADX71441 had an anxiolytic-like profile in the mouse marble burying test (minimum effective dose; MED 3 mg/kg) as well as in the elevated plus maze test in mice and rats (both MED 3 mg/kg). Also, in mice, acute administration of ADX71441 reduced visceral pain-associated behaviors in the acetic acid-induced writhing test. ADX71441 dose-dependently reduced time on rotarod in rats (MED 10 mg/kg) indicative of muscle-relaxant qualities. ADX71441 reduced locomotor activity in mice (10 mg/kg) and rats (3 mg/kg) after single dose; however, following sub-chronic administration in mice, 30 mg/kg ADX71441 was associated with normal locomotor activity. While acute administration of ADX71441 reduced body temperature in rats and mice (both MED 10 mg/kg), the effect in the former was transient, rapidly returning to normal levels despite high concentrations of the compound remaining in plasma. Thus, the GABAB PAM ADX71441 represents a valid therapeutic approach for development of novel treatment of anxiety, pain and spasticity.

Background and Purpose Glucose-dependent insulinotropic polypeptide (GIP) affects lipid, bone and glucose homeostasis. High-affinity ligands for the GIP receptor are needed to elucidate the physiological functions and pharmacological potential of GIP in vivo. GIP(1–30)NH2 is a naturally occurring truncation of GIP(1–42). Here, we have characterized eight N-terminal truncations of human GIP(1–30)NH2. Experimental Approach COS-7 cells were transiently transfected with human GIP receptors and assessed for cAMP accumulation upon ligand stimulation or competition binding with 125I-labelled GIP(1–42), GIP(1–30)NH2, GIP(2–30)NH2 or GIP(3–30)NH2. Key Results GIP(1–30)NH2 displaced 125I-GIP(1–42) as effectively as GIP(1–42) (Ki 0.75 nM), whereas the eight truncations displayed lower affinities (Ki 2.3–347 nM) with highest affinities for GIP(3–30)NH2 and GIP(5–30)NH2 (5–30)NH2. Only GIP(1–30)NH2 (Emax 100% of GIP(1–42)) and GIP(2–30)NH2 (Emax 20%) were agonists. GIP(2- to 9–30)NH2 displayed antagonism (IC50 12–450 nM) and Schild plot analyses identified GIP(3–30)NH2 and GIP(5–30)NH2 as competitive antagonists (Ki 15 nM). GIP(3–30) NH2 was a 26-fold more potent antagonist than GIP(3–42). Binding studies with agonist (125I-GIP(1–30)NH2), partial agonist (125I-GIP(2–30)NH2) and competitive antagonist (125I-GIP(3–30)NH2) revealed distinct receptor conformations for these three ligand classes. Conclusions and Implications The N-terminus is crucial for GIP agonist activity. Removal of the C-terminus of the endogenous GIP(3–42) creates another naturally occurring, more potent, antagonist GIP(3–30)NH2, which like GIP(5–30)NH2, was a high-affinity competitive antagonist. These peptides may be suitable tools for basic GIP research and future pharmacological interventions.

Linked article This article is commented on by Michel, M. C., pp. 429-430 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.13379. Background and purpose Mirabegron is the first β3 -adrenoceptor agonist approved for treatment of overactive bladder syndrome. This study aimed to investigate the effects of β3 -adrenoceptor agonist mirabegron in mouse urethra. The possibility that mirabegron also exerts α1 -adrenoceptor antagonism was also tested in rat smooth muscle preparations presenting α1A - (vas deferens and prostate), α1D - (aorta) and α1B -adrenoceptors (spleen). Experimental approach Functional assays were carried out in mouse and rat isolated tissues. Competition assays for the specific binding of [(3) H]prazosin to membrane preparations of HEK-293 cells expressing each of the human α1 -adrenoceptors, as well as β-adrenoceptor mRNA expression and cyclic AMP measurements in mouse urethra, were performed. Key results Mirabegron produced concentration-dependent urethral relaxations that were shifted to the right by the selective β3 -adrenoceptor antagonist L-748,337 but unaffected by β1 - and β2 -adrenoceptor antagonists (atenolol and ICI-118,551 respectively). Mirabegron-induced relaxations were enhanced by the PDE4 inhibitor rolipram, and the agonist stimulated cAMP synthesis. Mirabegron also produced rightward shifts in urethral contractions induced by the α1 -adrenoceptor agonist phenylephrine. Schild regression analysis revealed that mirabegron behaves as a competitive antagonist of α1 -adrenoceptors in urethra, vas deferens and prostate (α1A -adrenoceptor, pA2 ≅ 5.6) and aorta (α1D -adrenoceptor, pA2 ≅ 5.4) but not in spleen (α1B -adrenoceptor). The affinities estimated for mirabegron in functional assays were consistent with those estimated in radioligand binding with human recombinant α1A - and α1D -adrenoceptors (pKi ≅ 6.0). Conclusion and implications The effects of mirabegron in urethral smooth muscle are the result of β3 -adrenoceptor agonism together with α1A and α1D -adrenoceptor antagonism.

Toll-like receptor 2 (TLR2) induces early inflammatory responses to pathogen and damage-associated molecular patterns trough heterodimerization with either TLR1 or TLR6. Since overstimulation of TLR2 signaling is linked to several inflammatory and metabolic diseases, TLR2 antagonists may provide therapeutic benefits for the control of inflammatory conditions. We present virtual screening for the identification of novel TLR2 modulators, which combines analyses of known ligand sets with structure-based approaches. The 13 identified compounds were pharmacologically characterized in HEK293-hTLR2 cells, THP-1 macrophages and peripheral blood mononuclear cells for their ability to inhibit TLR2-mediated responses. Four out of 13 selected compounds show concentration-dependent activity, representing a hit rate of 31%. The most active compound is the pyrogallol derivative MMG-11 that inhibits both TLR2/1 and TLR2/6 signaling and shows a higher potency than the previously discovered CU-CPT22. Concentration ratio analysis identified both compounds as competitive antagonists of Pam3CSK4- and Pam2CSK4-induced responses. Schild plot analysis yielded apparent pA2 values of 5.73 and 6.15 (TLR2/1), and 5.80 and 6.65 (TLR2/6) for CU-CPT22 and MMG-11, respectively. MMG-11 neither shows cellular toxicity nor interference with signaling induced by other TLR agonists, IL-1β or TNF. Taken together, we demonstrate that MMG-11 is a potent and selective TLR2 antagonist with low cytotoxicity rendering it a promising pharmacological tool for the investigation of TLR signaling and a suitable lead structure for further chemical optimization.

Proguanil is an antimalarial prodrug that is metabolized to 4-chlorophenyl-1-biguanide (CPB) and the active metabolite cycloguanil (CG). These compounds are structurally related to meta-chlorophenyl biguanide (mCPBG), a 5-hydroxytryptamine 3 (5-HT3) receptor agonist. Here we examine the effects of proguanil and its metabolites on the electrophysiology and ligand-binding properties of human 5-HT3A receptors expressed in Xenopus oocytes and human embryonic kidney 293 cells, respectively. 5-HT3 receptor responses were reversibly inhibited by proguanil, with an IC50 of 1.81 μM. Competitive antagonism was shown by a lack of voltage-dependence, Schild plot (Kb = 1.70 μM), and radioligand competition (Ki = 2.61 μM) with the 5-HT3 receptor antagonist [(3)H]granisetron. Kinetic measurements (kon = 4.0 × 10(4) M(-1) s(-1) ; koff = 0.23 s(-1)) were consistent with a simple bimolecular reaction scheme with a Kb of 4.35 μM. The metabolites CG and CPB similarly inhibited 5-HT3 receptors as assessed by IC50 (1.48 and 4.36 μM, respectively), Schild plot (Kb = 2.97 and 11.4 μM), and radioligand competition (Ki = 4.89 and 0.41 μM). At higher concentrations, CPB was a partial agonist (EC50 = 14.1 μM; I/Imax = 0.013). These results demonstrate that proguanil competitively inhibits 5-HT3 receptors, with an IC50 that exceeds whole-blood concentrations following its oral administration. They may therefore be responsible for the occasional gastrointestinal side effects, nausea, and vomiting reported following its use. Clinical development of related compounds should therefore consider effects at 5-HT3 receptors as an early indication of possible unwanted gastrointestinal side effects.

Background The aim of our study was (1) the pharmacological characterization of EP 3 receptors in human pulmonary arteries and (2) the examination of the potential involvement of these receptors in the regulation of neurogenic tachycardia in pithed rats. L-826266 served as the EP3 receptor antagonist. Methods Experiments were performed on isolated human pulmonary arteries and pithed rats. Results The prostanoid EP 1 /EP 3 receptor agonist sulprostone (1 nM – 100 μM) concentration-dependently contracted isolated human pulmonary arteries (pEC 50 , 6.88 ± 0.10). The EP 1 receptor antagonist SC 19920 (100 μM) did not affect the vasoconstriction induced by sulprostone, the TP receptor antagonist sulotroban (10 μM) only slightly attenuated the effects elicited by sulprostone > 3 μM, whereas L-826266 (10 μM) shifted its concentration-response curve to the right (apparent pA 2 value 6.18; incubation time 0.5 h). In rings exposed to L-826266 (0.1,1 or 10 μM) for 3 h, a concentration-dependent inhibitory effect against the sulprostone-induced vasoconstriction was obtained, yielding a Schild plot-based pA 2 value of 7.39. In pithed rats, sulprostone (10 – 1,000 nmol/kg), but not the IP/EP 1 receptor agonist iloprost (1-100 nmol/kg), inhibited the electrically evoked increase in heart rate (HR) dosedependently, maximally by at least 80%. L-826266 (3 μmol/kg) did not affect basal HR and diastolic blood pressure, but reduced the inhibitory effect of sulprostone 1,000 nmol/kg by about 20%. Conclusion EP 3 receptors (1) located postsynaptically strongly contract human pulmonary arteries and (2) located presynaptically on sympathetic nerve fibers supplying the heart of pithed rats strongly inhibit the neurogenic tachycardia.

BACKGROUND AND PURPOSE Developmental switches in NMDA receptor subunit expression have been inferred from studies of GluN2 expression levels, changes in kinetics of glutamatergic synaptic currents and sensitivity of NMDA receptor-mediated currents to selective GluN2B antagonists. Here we use TCN 213, a novel GluN2A-selective antagonist to identify the presence of this subunit in functional NMDA receptors in developing cortical neurones. EXPERIMENTAL APPROACH Two-electrode voltage-clamp (TEVC) recordings were made from Xenopus laevis oocytes to determine the pharmacological activity of TCN 213 at recombinant NMDA receptors. TCN 213 antagonism was studied in cultures of primary cortical neurones, assessing the NMDA receptor dependency of NMDA-induced excitotoxicity and monitoring developmental switches in NMDA receptor subunit composition. KEY RESULTS TCN 213 antagonism of GluN1/GluN2A NMDA receptors was dependent on glycine but independent of glutamate concentrations in external recording solutions. Antagonism by TCN 213 was surmountable and gave a Schild plot with unity slope. TCN 213 block of GluN1/GluN2B NMDA receptor-mediated currents was negligible. In cortical neurones, at a early developmental stage predominantly expressing GluN2B-containing NMDA receptors, TCN 213 failed to antagonize NMDA receptor-mediated currents or to prevent GluN2B-dependent, NMDA-induced excitoxicity. In older cultures (DIV 14) or in neurones transfected with GluN2A subunits, TCN 213 antagonized NMDA-evoked currents. Block by TCN 213 of NMDA currents inversely correlated with block by ifenprodil, a selective GluN2B antagonist. CONCLUSIONS AND IMPLICATIONS TCN 213 selectively blocked GluN1/GluN2A over GluN1/GluN2B NMDA receptors allowing direct dissection of functional NMDA receptors and pharmacological profiling of developmental changes in native NMDA receptor subunit composition.

BACKGROUND AND PURPOSE Nicotinic acetylcholine receptors (nAChRs) containing α6β2 subunits expressed by dopamine neurons regulate nicotine-evoked dopamine release. Previous results show that the α6β2* nAChR antagonist, N,N′-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB) inhibits nicotine-evoked dopamine release from dorsal striatum and decreases nicotine self-administration in rats. However, overt toxicity emerged with repeated bPiDDB treatment. The current study evaluated the preclinical pharmacology of a bPiDDB analogue. EXPERIMENTAL APPROACH The C10 analogue of bPiDDB, N,N-decane-1,10-diyl-bis-3-picolinium diiodide (bPiDI), was evaluated preclinically for nAChR antagonist activity. KEY RESULTS bPiDI inhibits nicotine-evoked [3H]dopamine overflow (IC50 = 150 nM, Imax = 58%) from rat striatal slices. Schild analysis revealed a rightward shift in the nicotine concentration–response curve and surmountability with increasing nicotine concentration; however, the Schild regression slope differed significantly from 1.0, indicating surmountable allosteric inhibition. Co-exposure of maximally inhibitory concentrations of bPiDI (1 µM) and the α6β2* nAChR antagonist α-conotoxin MII (1 nM) produced inhibition not different from either antagonist alone, indicating that bPiDI acts at α6β2* nAChRs. Nicotine treatment (0.4 mg·kg−1·day−1, 10 days) increased more than 100-fold the potency of bPiDI (IC50 = 1.45 nM) to inhibit nicotine-evoked dopamine release. Acute treatment with bPiDI (1.94–5.83 µmol·kg−1, s.c.) specifically reduced nicotine self-administration relative to responding for food. Across seven daily treatments, bPiDI decreased nicotine self-administration; however, tolerance developed to the acute decrease in food-maintained responding. No observable body weight loss or lethargy was observed with repeated bPiDI. CONCLUSIONS AND IMPLICATIONS These results are consistent with the hypothesis that α6β2* nAChR antagonists have potential for development as pharmacotherapies for tobacco smoking cessation.

BACKGROUND AND PURPOSE Muscarinic and adrenergic G protein-coupled receptors (GPCRs) are the targets of rare peptide toxins isolated from snake or cone snail venoms. We used a screen to identify novel toxins from Dendroaspis angusticeps targeting aminergic GPCRs. These toxins may offer new candidates for the development of new tools and drugs. EXPERIMENTAL APPROACH In binding experiments with 3H-rauwolscine, we studied the interactions of green mamba venom fractions with α2-adrenoceptors from rat brain synaptosomes. We isolated, sequenced and chemically synthesized a novel peptide, ρ-Da1b. This peptide was pharmacologically characterized using binding experiments and functional tests on human α2-adrenoceptors expressed in mammalian cells. KEY RESULTS ρ-Da1b, a 66-amino acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. Its synthetic homologue inhibited 80% of 3H-rauwolscine binding to the three α2-adrenoceptor subtypes, with an affinity between 14 and 73 nM and Hill slopes close to unity. Functional experiments on α2A-adrenoceptor demonstrated that ρ-Da1b is an antagonist, shifting adrenaline activation curves to the right. Schild regression revealed slopes of 0.97 and 0.67 and pA2 values of 5.93 and 5.32 for yohimbine and ρ-Da1b, respectively. CONCLUSIONS AND IMPLICATIONS ρ-Da1b is the first toxin identified to specifically interact with α2-adrenoceptors, extending the list of class A GPCRs sensitive to toxins. Additionally, its affinity and atypical mode of interaction open up the possibility of its use as a new pharmacological tool, in the study of the physiological roles of α2-adrenoceptor subtypes.

Prostaglandin E(2) (PGE(2)) and its analogue sulprostone inhibit noradrenaline and serotonin release in rodent tissues. We examined whether the receptor involved is blocked by the EP(3) antagonist L-826,266, whether such receptors also occur on central cholinergic neurones and retinal dopaminergic cells, whether PGE(2) is produced by the degradation of the endocannabinoid virodhamine and whether EP(3) receptor activation stimulates (35)S-GTPgammaS binding. Transmitter release was studied as electrically evoked tritium overflow in superfused tissues preincubated with (3)H-noradrenaline (which in the guinea pig retina labels dopaminergic cells), (3)H-serotonin or (3)H-choline. (35)S-GTPgammaS binding, a measure of G protein activation, was studied in mouse and guinea pig hippocampal membranes. L-826,266 antagonised the effect of sulprostone on noradrenaline release in the rat cortex, yielding a Schild plot-based pA(2) value of 7.56. Apparent pA(2) values in mouse cortex and rat vas deferens (noradrenaline release) and rat cortex (serotonin release) were 7.55, 7.87 and 7.67, respectively. PGE(2) did not affect acetylcholine release in rat brain and dopamine release in guinea pig retina. In seven mice tissues, noradrenaline release was inhibited by sulprostone but not affected by virodhamine. (35)S-GTPgammaS binding was not altered by sulprostone but stimulated by the cannabinoid agonist WIN 55,212-2. Prostaglandins of the E series inhibit monoamine release via EP(3) receptors at which L-826,266 is a competitive antagonist. EP(3) receptors that inhibit transmitter release are not present on central cholinergic neurones and retinal dopaminergic cells. Virodhamine is not converted to PGE(2). An EP(3) receptor model based on (35)S-GTPgammaS binding could not be identified.

Smoking is a significant health concern and strongly correlated with clinical depression. Depression is associated with decreased extracellular NE concentrations in brain. Smokers may be self-medicating and alleviating their depression through nicotine stimulated norepinephrine (NE) release. Several antidepressants inhibit NE transporter (NET) function, thereby augmenting extracellular NE concentrations. Antidepressants, such as bupropion, also inhibit nicotinic receptor (nAChR) function. The current study determined if a recently discovered novel nAChR antagonist, N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), inhibits nicotine-evoked NE release from superfused rat hippocampal slices. Previous studies determined that bPiDDB potently (IC(50)=2 nM) inhibits nicotine-evoked striatal [(3)H]dopamine (DA) release in vitro, nicotine-evoked DA release in nucleus accumbens in vivo, and nicotine self-administration in rats. In the current study, nicotine stimulated [(3)H]NE release from rat hippocampal slices (EC(50)=50 microM). bPiDDB inhibited (IC(50)=430 nM; I(max)=90%) [(3)H]NE release evoked by 30 microM nicotine. For comparison, the nonselective nAChR antagonist, mecamylamine, and the alpha7 antagonist, methyllycaconitine, also inhibited nicotine-evoked [(3)H]NE release (IC(50)=31 and 275 nM, respectively; I(max)=91% and 72%, respectively). Inhibition by bPiDDB and mecamylamine was not overcome by increasing nicotine concentrations; Schild regression slope was different from unity, consistent with allosteric inhibition. Thus, bPiDDB was 200-fold more potent inhibiting nAChRs mediating nicotine-evoked [(3)H]DA release from striatum than those mediating nicotine-evoked [(3)H]NE release from hippocampus.

Bis(7)-tacrine is a novel dimeric acetylcholinesterase inhibitor derived from tacrine that shows promise for the treatment of Alzheimer's disease. We have previously reported that bis(7)-tacrine inhibits GABA(A) receptors. In the present study we investigated the mechanism of bis(7)-tacrine inhibition of GABA(A) receptor function using whole-cell patch-clamp recording in cultured rat hippocampal neurons. Bis(7)-tacrine produced a gradual decline of GABA-activated current to a steady-state, but this was not an indication of use-dependence, as the gradually declining component could be eliminated by exposure to bis(7)-tacrine prior to GABA application. In addition, bis(7)-tacrine inhibition did not require the presence of agonist, and GABA-activated current recovered completely from inhibition by bis(7)-tacrine in the absence of agonist. The slow onset of inhibition by bis(7)-tacrine was not apparently due to an action at an intracellular site, as inclusion of 25 microM bis(7)-tacrine in the recording pipette did not alter inhibition by bis(7)-tacrine applied externally. Bis(7)-tacrine shifted the GABA concentration-response curve to the right in a parallel manner and the pA(2) value estimated from a Schild plot was 5.7. Bis(7)-tacrine increased the time constant of activation of GABA-gated ion channels without affecting the time constants of deactivation or desensitization. These results suggest that bis(7)-tacrine is a competitive GABA(A) receptor antagonist with slow onset and offset kinetics. The competitive inhibition of GABA receptors by bis(7)-tacrine could contribute to its ability to enhance memory.

We have recently shown that the postjunctional alpha(2)-adrenoceptor mediating contraction of porcine pulmonary veins is of the alpha(2C)-subtype. We could also demonstrate that alpha(1)-adrenoceptors might contribute to the contraction in that blood vessel. In the present study, we aimed at characterising the alpha(1)-adrenoceptor subtype(s) involved using pharmacological and molecular biological methods. In isolated rings of porcine pulmonary veins the typical alpha(1)-adrenoceptor agonist phenylephrine caused a concentration-dependent contraction that was inhibited by the alpha(1B)-adrenoceptor selective antagonists 1-[4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl]-2-[2-(isopropyl)-6-methoxyphenoxy]ethan-1-one (Rec15/2615; pA(2) 8.96+/-0.13) and 4-amino-2-[4-[1-(benzyloxycarbonyl)-2(S)-[[(1,1-dimethylethyl)amino]carbonyl]-piperazinyl]-6,7-dimethoxyquinazoline (L-765,314; pA(2) 7.22+/-0.05), as well as the alpha(1D)-adrenoceptor selective antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY7378; pA(2) 8.29+/-0.15, slope of the Schild plot 0.75+/-0.09, significantly different from unity, P

The postjunctional alpha-adrenoceptors in human long saphenous vein were characterized using alpha-adrenoceptor subtype selective agonists and antagonists. The order of potency for the alpha-adrenoceptor agonists used was: Clonidine (alpha 2) greater than BHT-920 (alpha 2) greater than naphazoline (alpha 2) greater than guanfacine (alpha 2) greater than cirazoline (alpha 1) greater than phenylephrine (alpha 1) greater than ST 587 (alpha 1) greater than BHT-933 (alpha 2). Clonidine had the same potency as noradrenaline (NA), but was 52 times more potent than phenylephrine (pEC50 7.09 and 5.37, respectively). Phenylephrine and guanfacine had intrinsic activities that did not differ from that of NA, whereas the intrinsic activities of the other agonists were significantly lower. The highly selective alpha 1-adrenoceptor antagonist prazosin in concentrations 10(-9)-10(-7) M was unable to cause a significant shift of the NA concentration response (cr) curve to the right. However, the alpha 2-adrenoceptor antagonists yohimbine and rauwolscine in concentrations 10(-8)-10(-6) M shifted the NA cr-curve towards higher concentrations. No concentration of any antagonist used significantly attenuated the maximum contraction. The slope of the regression line in the Schild plot differed significantly from unity for rauwolscine but not for yohimbine, and the pA2-values were 9.00 and 8.27, respectively. These results suggest that the contraction mediating alpha-adrenoceptors in the human saphenous vein are mainly of the alpha 2-type. However, the low slope value of the Schild plot for rauwolscine may indicate the presence of a small population of alpha 1-adrenoceptors.

Caffeic acid phenethyl ester (CAPE), which is derived from the propolis of honeybee hives, has been demonstrated to possess multiple pharmacological activities. In the present study, CAPE (6-25 microM) specifically inhibited collagen-induced platelet aggregation and the ATP release reaction in platelet suspensions.Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance (ESR) were used to assess the anti-platelet activity of CAPE. Fluorescein sodium-induced platelet thrombi in mesenteric microvessels of mice were used for an in vivo study.CAPE (15-100 microM) produced a concentration-related rightward displacement of the collagen concentration-response curve, and the Schild plot gave pA(2) and pA(10) values of 4.28+/-0.07 and 3.14+/-0.73, respectively, with a slope of -0.83+/-0.16, indicating specific antagonism. CAPE (25 microM) also inhibited platelet aggregation stimulated by the glycoprotein VI agonist, convulxin, and the alpha(2)beta(1) integrin agonist, aggretin. CAPE (25 microM) also markedly interfered with FITC-collagen binding to platelet membranes. CAPE (15 and 25 microM) concentration-dependently inhibited collagen-induced platelet activation accompanied by [Ca(+2)](i) mobilization, phosphoinositide breakdown, activation of protein kinase C and mitogen-activated protein kinases (i.e., ERK2, JNK, and p38 MAPK), Akt phosphorylation, and thromboxane A(2) formation. In the ESR study, CAPE (15 and 25 microM) markedly reduced hydroxyl radical (OH) formation in collagen-activated platelets. In an in vivo study, CAPE (5 mg/kg) significantly prolonged the latency in inducing platelet plug formation in mesenteric venules of mice.The most important findings of this study suggest that CAPE specifically inhibits collagen-induced platelet activation. Thus, CAPE treatment may represent a novel approach to lowering the risk of or improving function in thromboembolism-related disorders.

Although competitive antagonism experiments are critical tools in the classification of potential pharmacotherapies, no studies have quantitatively compared the potencies of 5-HT(2C) receptor antagonists using the Schild regression analysis in vivo.To evaluate the behavioral effects of 5-HT(2C) receptor agonists and antagonists, a series of nonselective 5-HT(2C) receptor antagonists, the 5-HT(2A/2C) receptor antagonist ketanserin, the 5-HT(2B) receptor antagonist SB 204,741, the 5-HT(2B/2C) receptor antagonist SB 200,646, and the peripherally acting 5-HT(2C) receptor antagonist RS102,221 were evaluated for their capacity to competitively antagonize the agonists MK212, mCPP, or BW723C86 in rats.Male Sprague-Dawley rats (N = 28) were trained to respond under a fixed ratio 10 schedule of food reinforcement. A multiple-trial, cumulative-dosing procedure was used to evaluate the capacity of the compounds to suppress response rates.MK212, mCPP, and the 5-HT(2B) receptor agonist BW723C86 dose-dependently decreased response rates. Only metergoline, mianserin, and methysergide produced a dose-dependent antagonism of the rate-decreasing effects of both mCPP and MK212. Apparent pA(2) analysis indicated that metergoline, mianserin, and methysergide were approximately equipotent as antagonists overall. Metergoline and mianserin failed to block the rate-decreasing effects of BW723C86. Ketanserin, SB 200,646, SB 204,741, and RS102,221 failed to block either mCPP or MK212, suggesting that 5-HT(2A), 5-HT(2B), or peripheral 5-HT(2C) receptors do not play a primary role in the rate-decreasing effects of these two agonists.Taken together, these antagonism profiles suggest that the agonists MK212 and mCPP produce their rate-decreasing effects through a combination of 5-HT receptors with the 5-HT(2C) receptor playing a prominent but not exclusive role.

Background and purpose: The aim was to characterize the recently discovered non-peptide antagonist MEN16132 at the mouse B2 receptor, relative to other antagonists. Experimental approach: [3H]-BK binding experiments used mouse lung and ileum tissue membranes and antagonist potency was measured in the isolated ileum contractility assay. Key results: Two BK binding sites resulted from saturation and homologous competition experiments. A role for the B1 receptor was excluded because of the poor affinity of B1 receptor ligands (pIC50 LF16-0687 (lung 8.9; ileum 8.8) > FR173657 (lung 8.6; ileum 8.2). BK homologous curves performed with lung membranes after treatment with the antagonist MEN16132 or Icatibant (10 nM) displayed only the low affinity site. The functional antagonism by MEN16132 (pA2 9.4) and Icatibant (pA2 9.1), towards BK (control EC50 6.1 nM) induced ileum contractions, was concentration-dependent and surmountable, but the Schild plot slope was less than unity. Conclusions and Implications: In mouse tissue, radiolabelled BK recognizes two binding sites and B2 receptor antagonists can compete only for the higher affinity one. The pharmacological profile of the novel non-peptide antagonist MEN16132 indicates that it exhibits subnanomolar affinity and potency for the mouse B2 receptor and is suitable for further characterization in in vivo pathophysiological models. British Journal of Pharmacology (2007) 150, 313–320. doi:10.1038/sj.bjp.0706995

The main aim of this study was to determine to what extent estimates of the receptor (b2:b1) selectivity of b-adrenoceptor antagonists obtained in vitro in the guinea-pig might reflect the tissue (trachea:heart) selectivity of the same antagonists in vivo in the same species. To achieve this aim in vitro experiments with guinea-pig isolated atria (rate) and intrinsic-tone trachea (relaxation) were carried out under optimal experimental conditions for adrenoceptor selectivity studies. The b-adrenoceptor agonists used in vitro were fenoterol, noradrenaline and isoprenaline and various b-adrenoceptor antagonists were investigated viz a-methylpropranolol, 4-BIP [(p)-l-(4-benzimidazoloxy)-3-isopropylamino-2-propanol], 5-BIP [(p)-l-(5-benzimidazoloxy-3-isopropyl amino-2-propanol] and IPS 339 [(t-butylamino-3-ol-2-propyl)oximino-9-fluorene]. All these antagonists had been claimed in prior studies as having some degree of b2-selectivity but the published data on which this conclusion was based were not obtained under conditions considered optimal for the determination of selectivity. Receptor selectivity estimates, calculated from the difference in pA2 values obtained on trachea, using fenoterol as agonist and on atria, using noradrenaline as agonist, were obtained for each antagonist and were 11.0 for a-methylpropranolol, 2.5 for 4-BIP and 4.9 for IPS 339; no estimate was calculated for 5-BIP. Experiments conducted with carbachol - contracted tracheal preparations from the same animals showed these to be unsuitable preparations for the estimation of receptor selectivity since conditions satisfying Schild plot theory could not be met.a-Methylpropranolol, being the most selective of these antagonist compounds, was selected for study in vivo. Before an in vivo estimate of its selectivity could be obtained an experimental model in the intact guinea-pig, which mirrored the in vitro responses, had to be developed. The experimental model chosen involved measuring heart rate, tracheal segment pressure and diastolic blood pressure responses to isoprenaline and other selective agonists in anaesthetized guinea-pigs and led to the following conclusions on methodology. Isoprenaline was selected as agonist since it gave short-acting, reproducible responses and could be given in a cummulative dose to enable several doses of the b-adrenoceptor antagonist to be investigated in any one guinea-pig. Endogenous catechol amines were depleted by the use of reserpine. Cardiovascular reflexes were reduced or inhibited by bilateral vagotomy. Responses were also obtained in the presence of the a-adrenoceptor antagonist phenoxybenzamine, to mimic as far as practicable conditions used in the in vitro experiments.Since only one b2-selective adrenoceptor antagonist, a-methylpropranolol, had been selected for the in vivo study, two other selective antagonists, for which satisfactory in vitro data were already available, were also studied in vivo. They were ICI 118,551 (erythro-DL-l-(7-methylindan-4-yloxy)-3-isopropylamino-butan-2-ol), having a b2-selectivity of 53.7 and atenolol, having a b1-selectivity of 27.8 (which is equivalent to a b2-selectivity of 0.036). Estimates of tissue (trachea:heart) selectivity in vivo were calculated from the difference in qin vivo pA2q values obtained on tracheal segment pressure and heart rate, using isoprenaline as agonist. These estimates were less than the receptor (b2:b1) selectivity estimates determined in vitro, i.e. they were 1.7 for a-methylpropranolol, 4.0 for ICI 118,551 and 0.23 for atenolol. a-Methylpropranolol selectivity increased marginally to 2.0 if the b2-selective agonist rimiterol was used in place of isoprenaline. Vascular:heart selectivity estimates were also obtained, but only for comparative purposes, and were 1.1 for a-methyl propranolol, 2.8 for ICI 118,551 and 0.12 for atenolol.It is concluded that the estimates of tissue (trachea:heart) selectivity of b-adrenoceptor antagonists obtained in vivo in guinea-pigs, usually with isoprenaline (non-selective) as agonist drug, follow the same relative trend as those obtained in vitro for the same tissues (isolated trachea, atrial rate) in the same species, but are quantitatively less. If selectivity in vitro is obtained using selective agonists instead of isoprenaline, then because of the possibility of heterogeneous b-adrenoceptor populations this difference is even more exaggerated. The use of selective agonists in vivo , to try and allow for the possibility that responses could be mediated by both subtypes of b-adrenoceptors, was hindered by technical problems due to, for example, vasoconstrictor actions (in the case of noradrenaline) and a prolonged duration of action of the agonists.

This study sets out to characterise calcitonin gene-related peptide (CGRP) receptors in human and porcine isolated proximal and distal coronary arteries using BIBN4096BS. Human (h)-alphaCGRP induced relaxations that were blocked by BIBN4096BS in all arteries studied. In contrast to the other vessels, the Schild plot slope in the human distal coronary artery segments (0.68 +/- 0.07) was significantly less than unity and BIBN4096BS potently blocked these responses (pK(b) (10 nM): 9.29 +/- 0.34, n = 5). In the same preparation, h-alphaCGRP(8-37) behaved as a weak antagonist of h-alphaCGRP-induced relaxations (pK(b) (3 microM): 6.28 +/- 0.17, n = 4), with also a Schild plot slope smaller than unity. The linear agonists, [ethylamide-Cys(2,7)]-h-alphaCGRP ([Cys(Et)(2,7)]-h-alphaCGRP) and [acetimidomethyl-Cys(2,7)]-h-alphaCGRP ([Cys(Acm)(2,7)]-h-alphaCGRP), had a high potency (pEC(50): 8.21 +/- 0.25 and 7.25 +/- 0.14, respectively), suggesting the presence of CGRP(2) receptors, while the potent blockade by BIBN4096BS (pK(b) (10 nM): 10.13 +/- 0.29 and 9.95 +/- 0.11, respectively) points to the presence of CGRP(1) receptors. Using RT-PCR, mRNAs encoding for the essential components for functional CGRP(1) receptors were demonstrated in both human proximal and distal coronary artery. Further, h-alphaCGRP (100 nM) increased cAMP levels, and this was attenuated by BIBN4096BS (1 microM). The above results demonstrate the presence of CGRP(1) receptors in all coronary artery segments investigated, but the human distal coronary artery segments seem to have an additional population of CGRP receptors not complying with the currently classified CGRP(1) or CGRP(2) receptors.

4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride (AC-42) is a selective agonist of the muscarinic M(1) receptor previously suggested to interact with an "ectopic" site on this receptor. However, the pharmacological properties of this site (i.e., whether it overlaps to any extent with the classic orthosteric site or represents a novel allosteric site) remain undetermined. In the present study, atropine or pirenzepine significantly inhibited the ability of either carbachol or AC-42 to stimulate inositol phosphate accumulation or intracellular calcium mobilization in Chinese hamster ovary (CHO) cells stably expressing the human M(1) receptor. However, the interaction between either of these antagonists and AC-42 was characterized by Schild slopes significantly less than unity. Increasing the concentrations of atropine revealed that the Schild regression was curvilinear, consistent with a negative allosteric interaction. More direct evidence for an allosteric mode of action of AC-42 was obtained in [(3)H]N-methylscopolamine ([(3)H]NMS) binding studies, in that both AC-42 and the prototypical modulator gallamine failed to fully inhibit specific [(3)H]NMS binding in a manner that was quantitatively described by an allosteric model applied to both modulator data sets. Furthermore, AC-42 and gallamine significantly retarded the rate of [(3)H]NMS dissociation from CHO-hM(1) cell membranes, conclusively demonstrating their ability to bind to a topographically distinct site to change M(1) receptor conformation. These data provide the first direct evidence that AC-42 is an allosteric agonist that activates M(1) receptors in the absence of the orthosteric agonist.

The smooth muscle relaxant properties of a mixture of the two triterpenoids cycloeuclalenol and cycloartenol (CC) isolated from Herissanthia tiubae (Malvaceae) were studied in several smooth muscle preparations. CC inhibited contractions induced by carbachol, histamine and KCl in the guinea-pig ileum, but no spasmolytic activity was found in guinea-pig trachea or rat aorta. In guinea-pig ileum, concentration-response curves to carbachol and CaCl (2) in high K(+) were shifted to the right by CC in a concentration-dependent manner with slopes of the Schild plot differing from the unity. IC(50) values were 3.4 +/- 0.8 x 10 (-5) M and 8.44 +/- 1.87 x 10 (-5) M for carbachol and CaCl(2), respectively. The phorbol ester TPA, which activates protein kinase C (PKC), potentiated contractions induced by submaximal concentrations of carbachol. This potentiation was inhibited by CC. Desensitization of PKC by TPA completely abolished the inhibition produced by CC on carbachol-induced contractions. Together, our results indicate that inhibition of PKC is involved in the spasmolytic effect of CC in the guinea-pig ileum.

Oxybutynin has been used for neurogenic bladder disorders in clinic and known to have anti-cholinergic and spasmolytic properties. Metabolite of oxybutynin, 4-ethylamino-2-butynyl(2-cyclohexyl-2-phenyl) glycolate (N-desethyloxybutynin: DEOB) has been known to have similar anti-cholinergic and spasmolytic properties. However, the effect of DEOB on the urinary bladder has not been clarified in situ. Therefore, in the present study, we studied the effect of DEOB on acetylcholine-induced urinary bladder contraction in comparison with oxybutynin in anesthetized dogs. Intravenously administered DEOB dose-dependently inhibited acetylcholine-induced contractions. Oxybutynin also showed similar efficacy. From the Schild plot, it was found that the slope of DEOB and oxybutynin were 0.78 (95% confidence limit: 0.45-1.11) and 1.49 (95% confidence limit: 0.91-2.08), respectively. The dose of DEOB or oxybutynin needed to shift the concentration-dependent curve of acetylcholine rightward to a two times higher dose was calculated. The doses of DEOB and oxybutynin were 6.4 micro g/kg (95% confidence limit: 1.7-12.8 micro g/kg) and 13.9 micro g/kg (95% confidence limit: 6.3-24.5 micro g/kg), respectively. From the above results, it was found that DEOB has the same anti-cholinergic property as oxybutynin and that its activity was almost equipotent to that of oxybutynin. Therefore, DEOB was suggested to play an important role during oxybutynin therapy for neurogenic bladder disorder.

The 'Schild regression' method is based on the principle of assessing the rightward shift of agonist dose-effect curves in the presence of different doses/concentrations of the respective receptor antagonist and presenting their relationship in a double log plot (i.e. the 'Schild plot'). The original method was developed to quantitatively characterize antagonistic drugs in experimental pharmacology. The method was adopted for evaluation of various AT1 antagonists in humans utilizing (human) angiotensin II as the agonist. Angiotensin II (Ang II) in continuous intravenous dose-incremental administration resulted in a clearly dose-dependent increase in blood pressure. All AT1 antagonists tested after oral administration yielded concentration-dependent rightward shifts of those Ang II dose-effect curves that were quantified as dose ratio (DR). DR minus 1 (DR-1) enabled the assessment of antagonist time kinetics in humans and a quantitatively precise determination of the half-life of antagonism in vivo. Schild plots allowed for assessment of apparent Ki doses indicative of a twofold rightward shift of the Ang II effect, thus providing the means for a rational comparison of the pharmacological potency of many of these compounds, where the Ki doses obtained at 24 h after administration were in the range of 'therapeutic' doses. Schild plots of a variety of substances showed linear relations independent of whether the blockade was deemed surmountable or not. It is therefore assumed that this property does not play a role at clinical doses/concentrations. Slopes slightly below 1 in the Schild plots of all tested antagonists point to a second 'counterregulatory' vasodilatory mechanism of action of Ang II which becomes apparent with AT1 blockade in conditions of high doses/concentrations of Ang II. Concentration vs. effect relationships indicate that if assessed at the same degree of direct vascular antagonism, other effects, such as increase in plasma renin activity, may be present to a varying degree with different antagonists. Thus for irbesartan, the potency to stimulate renin release was found to be at least twice that of candesartan. These observations should stimulate further research into the relevance of these dynamic differences between the various compounds. Thus, methodologies relying on fundamental principles of experimental pharmacology can provide the clinical pharmacologist with powerful tools to measure accurately degree of antagonism and time kinetics and to investigate the nature of receptor antagonism in humans.

1. In PC12 cells, adenine nucleotides inhibit voltage-activated Ca(2+) currents and adenylyl cyclase activity, and the latter effect was reported to involve P2Y(12) receptors. To investigate whether these two effects are mediated by one P2Y receptor subtype, we used the antithrombotic agents 2-methylthio-AMP (2-MeSAMP) and N(6)-(2-methyl-thioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP (AR-C69931MX). 2. ADP reduced A(2A) receptor-dependent cyclic AMP synthesis with half maximal effects at 0.1-0.17 micro M. In the presence of 30 micro M 2-MeSAMP or 100 nM AR-C69931MX, concentration response curves were shifted to the right by factors of 39 and 30, indicative of pA(2) values of 6.1 and 8.5, respectively. 3. The inhibition of Ca(2+) currents by ADP was attenuated by 10-1000 nM AR-C69931MX and by 3-300 micro M 2-MeSAMP. ADP reinhibited Ca(2+) currents after removal of 2-MeSAMP within less than 15 s, but required 2 min to do so after removal of AR-C69931MX. 4. ADP inhibited Ca(2+) currents with half maximal effects at 5-20 micro M. AR-C69931MX (10-100 nM) displaced concentration response curves to the right, and the resulting Schild plot showed a slope of 1.09 and an estimated pK(B) value of 8.7. Similarly, 10-100 micro M 2-MeSAMP also caused rightward shifts resulting in a Schild plot with a slope of 0.95 and an estimated pK(B) of 5.4. 5. The inhibition of Ca(2+) currents by 2-methylthio-ADP and ADPbetaS was also antagonized by AR-C69931MX, which (at 30 nM) caused a rightward shift of the concentration response curve for ADPbetaS by a factor of 3.8, indicative of a pA(2) value of 8.1. 6. These results show that antithrombotic drugs antagonize the inhibition of neuronal Ca(2+) channels by adenine nucleotides, which suggests that this effect is mediated by P2Y(12) receptors.

We compared the effects of CL-316243, a selective β3-adrenoceptor agonist, and CGP-12177A, a nonconventional partial β-adrenoceptor agonist, on the KCl-induced contraction in the isolated canine ureter. CL-316243 concentration dependently relaxed the ureteral contraction, the pD2 value being 7.75 ± 0.11. This relaxation was competitively antagonized by the selective β3-adrenoceptor antagonist SR58894A and by the nonselective β-adrenoceptor antagonist bupranolol, their pA2 values being 7.08 ± 0.08 and 6.43 ± 0.09, respectively. CGP-12177A concentration dependently reduced the KCl-induced contraction, the pD2 value being 6.30 ± 0.25. Even at 1 × 10–5 mol/l, CGP-20712A (a selective β1- adrenoceptor antagonist) did not shift the concentration-response curves for CL-316243 or CGP-12177A. SR58894A did not induce a parallel rightward shift in the concentration-response curve for CGP-12177A, but bupranolol did produce such a shift, pA2 and slope values in the Schild plot being 7.15 ± 0.77 and 0.60 ± 0.15, respectively. Hence, the competition characteristics for SR58894A and bupranolol differed between the CL- 316243-induced and CGP-12177A-induced relaxations. Our results suggest that CGP-12177A produces ureteral relaxation in the dog via an atypical β-adrenoceptor (possibly, an atypical site/state of the β3-adrenoceptor) as well as via the typical β3-adrenoceptor.

Aims To investigate the role of β-adrenoceptor subtypes in mediating relaxation of the pig bladder trigonal muscle in vitro. Materials and Methods Longitudinal strips of trigonal muscle were isolated, and the mucosa and serosa removed. Tissues were precontracted with KCl, and β-adrenoceptor agonists (isoprenaline or salbutamol) were added cumulatively, and concentration–relaxation curves (CRCs) were obtained. CRCs to agonists were obtained in the absence and presence of antagonists and antagonist affinity values were calculated. Results Isoprenaline (non-selective β-agonist) relaxed with high potency (pEC50 = 7.2). Propranolol antagonized CRCs to isoprenaline with a high affinity (apparent pKB = 8.8), but the Schild plot had a slope significantly (P

1. The natural product willardiine is a selective AMPA receptor agonist. We report that an N(3)-substituted analogue of willardiine, (S)-3-(4-carboxybenzyl)willardiine 3-CBW, antagonizes AMPA and kainate receptors expressed on motoneurones and dorsal root C-fibres, respectively. 2. Reduction of the fast component of the dorsal root-evoked ventral root potential (fDR-VRP) has been used as a novel method to compare AMPA receptor antagonists. 3-CBW, NBQX and GYKI53655 depressed the fDR-VRP with IC(50) values of 10.3+/-2.4, 0.214+/-0.043 and 4.03+/-0.31 micro M, respectively. That 3-CBW depressed the fDR-VRP by acting at AMPA and not metabotropic glutamate receptors was demonstrated by the lack of effect of LY341495 (100 micro M). 3. The Schild plot for antagonism of responses to (S)-5-fluorowillardiine on motoneurones by 3-CBW had a slope of 1.11+/-0.13 giving a pA(2) value of 4.48. The Schild plot for antagonism of kainate responses on the dorsal root by 3-CBW had a slope of 1.05+/-0.05 giving a pA(2) value of 4.96. 4. On neonatal rat motoneurones 3-CBW (200 micro M) almost completely abolished responses to AMPA while responses to NMDA, kainate and DHPG were 101.6+/-11.6%, 39.4+/-5.8% and 110.5+/-9.0% of control, respectively. 3-CBW can therefore be used to isolate kainate receptor responses from those mediated by AMPA receptors. 5 3-CBW antagonized kainate-induced responses on dorsal root C-fibres with a pA(2) value of 4.96 whereas kainate receptor mediated responses (isolated by including GYKI53655 in the medium) on motoneurones were not completely blocked by 200 micro M 3-CBW, substantiating evidence that kainate receptors on neonatal rat motoneurones differ from those on dorsal root C-fibres.

Schild regressions for the selective AT 1 and AT 2 receptor antagonists, losartan and PD123319 ( S -[+]-1-[(4-dimethylamino]-3-methylphenyl)methyl]-5-[diphenylacetyl]-4,5,6,7-tetrahydro-1 H -imidazol[4,5- c ]pyridine-6-carboxilic acid), respectively, were calculated to analyze the heterogeneity of receptor populations in the rat anococcygeus muscle. For a one-receptor system, the Schild regression has a slope of unity and an intercept of K B for competitive antagonists. However, in a two-receptor system, a deviation from the single-receptor plot will occur. This is predicated on the assumption that the secondary receptor is less sensitive to the antagonist than the primary receptor. Results showed that the Schild regression for losartan did not produce a slope of unity, and PD123319 did not produce any effect. However, tissue incubation with losartan plus PD123319 resulted in a Schild regression that has a slope of unity and a p K B of 9.32. In the presence of prazosin, an α 1 -adrenoceptor antagonist, losartan did not produce any effect. Conversely, PD123319 enhanced the angiotensin II (Ang II)-induced contraction in a concentration-dependent fashion, suggesting an inhibitory AT 2 -mediated effect. This effect was confirmed with assays that showed a relaxant response induced by Ang II on precontracted tissues incubated with prazosin. PD123319 and N G -nitro-l-arginine methyl ester [nitric-oxide (NO) synthase inhibitor)] markedly inhibited the relaxant response of Ang II. In contrast, losartan did not produce any significant effect. Consequently, results show that the mechanism underlying the AT 2 -mediated effect is highly dependent on NO generation. Results indicate the presence of a heterogeneous angiotensin receptor population in the rat anococcygeus muscle following a negative cross-talk relationship between the AT 1 and AT 2 subtypes.

1. The ability of the CGRP antagonist BIBN4096BS to antagonize CGRP and adrenomedullin has been investigated on cell lines endogenously expressing receptors of known composition. 2. On human SK-N-MC cells (expressing human calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1)), BIBN4096BS had a pA 2 of 9.95 although the slope of the Schild plot (1.37±0.16) was significantly greater than 1. 3. On rat L6 cells (expressing rat CRLR and RAMP1), BIBN4096BS had a pA 2 of 9.25 and a Schild slope of 0.89±0.05, significantly less than 1. 4. On human Colony (Col) 29 cells, CGRP 8-37 had a significantly lower pA 2 than on SK-N-MC cells (7.34±0.19 (n=7) compared to 8.35±0.18, (n=6)). BIBN4096BS had a pA 2 of 9.98 and a Schild plot slope of 0.86±0.19 that was not significantly different from 1. At concentrations in excess of 3 nM, it was less potent on Col 29 cells than on SK-N-MC cells. 5. On Rat 2 cells, expressing rat CRLR and RAMP2, BIBN4096BS was unable to antagonize adrenomedullin at concentrations up to 10 μM. CGRP 8-37 had a pA 2 of 6.72 against adrenomedullin. 6. BIBN4096BS shows selectivity for the human CRLR/RAMP1 combination compared to the rat counterpart. It can discriminate between the CRLR/RAMP1 receptor expressed on SK-N-MC cells and the CGRP-responsive receptor expressed by the Col 29 cells used in this study. Its slow kinetics may explain its apparent 'non-competive' behaviour. At concentrations of up to 10 μM, it has no antagonist actions at the adrenomedullin, CRLR/RAMP2 receptor, unlike CGRP 8-37.

1 b-adrenoceptors mediate relaxation of bladder detrusor smooth muscle. This study investigates the contribution of b3-adrenoceptors to relaxation of the pig urinary bladder. 2 Cell membranes were prepared from detrusor muscle of the pig bladder dome and competition experiments with [ 3 H]-dihydroalprenolol (DHA), a non-selective b-adrenoceptor antagonist was used as a specific radioligand to determine the presence of b-adrenoceptor subtypes. In functional experiments, isolated detrusor muscle strips were used to determine the potency of agonists and the aAnity of antagonists. 3 In competition binding experiments, CGP20712A (b1-adrenoceptor selective) displaced [ 3 H]DHA from a single binding site with a low aAnity. In contrast, displacement data for ICI 118551 (b2-adrenoceptor antagonist) and SR59230A (b3-adrenoceptor antagonist) best fitted a two-site model suggesting a predominant (70%) population of b3-adrenoceptors. 4 In functional studies, isoprenaline and salbutamol (b2-adrenoceptor agonist) relaxed KCl precontracted muscle strips with high potency (pEC50 7.7 and 7.2, respectively), whilst CGP12177 and BRL37344 (b3-adrenoceptor agonists) had low potency and were partial agonists. CGP20712A and atenolol (b1-adrenoceptor antagonists) antagonised responses with a low aAnity. ICI118551 antagonized responses to isoprenaline and salbutamol with a high aAnity (pKB=7.8 and 8.7, respectively), but the Schild slopes were low suggesting that responses were mediated by more than one b-adrenoceptor. The Schild plot for SR59230A was biphasic, apparent pKB values for 3‐10 nM SR59230A being 8.6 and those for 30 nM‐1mM being 7.7. 5 These data suggest that b3-adrenoceptors are the predominant b-adrenoceptor subtype present in the pig bladder and that b-adrenoceptor mediated responses of this tissue are mediated via both the b2- and b3-adrenoceptor subtypes. British Journal of Pharmacology (2002) 135, 129‐134

This chapter discusses the blockade of agonist-induced response through interaction with receptors. Antagonism can be classified operationally in terms of the effects of antagonists on agonist dose-response curves and mechanistically in terms of the molecular effects of the antagonist on the receptor protein. The interference of agonist-induced response can take different forms in terms of the effects on agonist dose-response curves. Specifically, concentration-dependent antagonism can be saturable or apparently unsaturable. The antagonism can be surmountable (dextral displacement of the dose-response curve with no diminution of maxima) or insurmountable (depression of the maximal agonist response). Antagonism of receptors can produce many patterns of concentration-response curves for agonists, including concentration-dependent surmountable antagonism, surmountable antagonism that comes to a maximal limit, depression of dose-response curves with no dextral displacement, and dextral displacement before depression of maximal response in systems with a receptor reserve for the agonist. These patterns are recognized as behaviors of antagonists in different systems and not necessarily characteristics of the molecular nature of the antagonism (i.e., more than one molecular mechanism can produce the same behavior of the concentration-response curves). Therefore, it is important to discover the molecular mechanism of the antagonism and not just describe the antagonistic behavior, as the latter can change with experimental conditions. Kinetic factors can cause some antagonists to produce surmountable antagonism in some systems and insurmountable antagonism in others.

Introduction: The Cheng–Prusoff equation (1973) is often applied to the determination of equilibrium dissociation constant ( K B ) of a competitive antagonist when the IC 50 value is available. The purpose of this study is to illustrate that the slope function ( K ) of an agonist concentration–response curve is critical to the determination of K B values. Methods: The article describes new equations, which incorporate the slope function, consequently yielding more accurate estimation of K B values for antagonists, and tests them using simulated data. The value of K B was calculated according to the following new power equation: K B =IC 50 /(1+ A K / K P )=IC 50 /[1+( A /EC 50 ) K ], where IC 50 is the concentration of the antagonist producing 50% inhibition, A is the concentration of the agonist against which the IC 50 is being determined and K P is the apparent equilibrium dissociation constant of the agonist. Results: The new equation is the same as the Cheng–Prusoff equation when the slope function K is exactly unity. Application of the equation avoids errors inherent in the use of the Cheng–Prusoff equation when the slope function of the agonist concentration–response curve deviates from unity. The new equation was applicable to slope functions less than, equal to or greater than unity. All inhibition curves have a negative slope function of 1, indicating that there is only one single receptor population even though different slope functions of agonist concentration–response curves are involved. The importance of the power function in the Schild plot is illustrated by using the equation: log ( x K −1)=log B −log K B , where x is the concentration ratio and B is the concentration of the antagonist. Discussion: This investigation illustrates the application of six power equations for accurate estimation of K B values covering situations with different slope functions of the agonist concentration–response curves.

GYKI-46903 [(+)-(5S,6R)-4-(4-fluorophenyl)-6-propionyloxy-1-aza-bicyclo[3.3.1]-non-3-ene-hydrochloride], a cognition enhancer identified as a non-competitive antagonist of 5-HT3receptors in isolated guinea-pig ileum, was investigated for allosteric action at 5-HT3 receptors in rat cortical membranes by using [3H]granisetron. Equilibrium and kinetic protocols were applied and the competitive antagonist granisetron was included as a negative control. In competition studies, both granisetron and GYKI-46 903 displaced the radioligand with K(i) values of 0.20 +/- 0.02 and 79.84 +/- 0.28 nM, respectively. The inhibition curve for GYKI-46 903 resulted in a Hill slope significantly greater than unity ( 1.37 +/- 0.11), whereas the slope for granisetron was 0.88 +/- 0.08, not different from unity. These results indicate non-competitive and competitive interactions, respectively. Scatchard analysis yielded a linear plot, suggesting a single population of binding sites with a Kd of 0.13 +/- 0.01 nM and a Bmax) of 13.15 +/- 0.34 fmol per mg of protein. Scatchard plots obtained in the absence and presence of granisetron (0.1-3 nM) or GYKI-46 903 (30-1000 nM) revealed a concentration-dependent increase in Kd values by either of these compounds. Granisetron left the Bmax unchanged, but there was a significant increase in the Bmax by GYKI-46 903, which could point to an atypical allosteric interaction. The Schild plot derived from the Kd shifts induced by granisetron was linear with a slope of 1.02, not different from unity, as expected from a competitive interaction. The Schild regression for GYKI-46 903 was linear with a slope of 1.20, deviating significantly from unity, which may also indicate an allosteric interaction. Both the association and dissociation curves of [3H]granisetron were monoexponential. The dissociation rate constant (K(-1)) and the association rate constant (K(+1)) were 0.32 +/- 0.01 min(-1) and 1.15 min(-1) x nM(-1), respectively. The dissociation driven by an excess concentration of ondansetron ( 1 microM) in the absence and presence of granisetron (0.1-3 nM) or GYKI-46 903 (30-10 000 nM) was not influenced by the compounds under study, as compared with the control, indicating the lack of an allosteric effect on the dissociation. Summing up, the binding profile of GYKI-46 903 may reflect a mixed type of action, including a negative allosteric interaction.

Rationale A quantitative technique was used to compare the pharmacological potency in healthy volunteers of angiotensin II receptor antagonists (AIIA): candesartan cilexetil, losartan, irbesartan, valsartan, and telmisartan. Methods In a randomised, double-blind, parallel-group (4x12 subjects) study, single oral doses of candesartan cilexetil 4, 8 and 16 mg, losartan potassium 25, 50 and 100 mg, valsartan 40, 80 and 160 mg, and irbesartan 75, 150 and 300 mg were administered on three consecutive days. Telmisartan 20, 40 and 80 mg was similarly evaluated in 12 volunteers in an open amendment. Angiotensin II (Ang II) antagonistic effects were determined in vivo from rightward shifts in Ang II dose-response curves for diastolic blood pressure (BP) and dose ratios were calculated. Apparent Ki-doses, i.e. doses (in mg) required to induce a two-fold shift in Ang II dose-response curves (equivalent to approx. 50% blockade of receptors) were determined, using Schild regression analysis. Results All treatments dose-dependently attenuated increases in diastolic BP induced by infusion of exogenous Ang II. Candesartan cilexetil appeared to have a more pronounced increase in effect following cumulative dosing. At 24 hours, apparent K i-doses were: candesartan cilexetil 6 mg, irbesartan 123 mg, valsartan 93.5 mg, and telmisartan 54 mg. It was not possible to determine an apparent Ki-dose for losartan at 24 hours. Conclusion Consistent with results from experimental pharmacology, candesartan cilexetil displayed the highest pharmacological potency (i.e. antagonistic activity per mg substance) of the AIIAs tested. Apparent Ki-doses at 24 hours were within the dose range recommended for clinical use in patients with hypertension.

The P(2)-purinoceptor antagonist, suramin, was used to investigate the possible involvement of adenosine 5'-triphosphate (ATP) in the inhibitory non-adrenergic non-cholinergic (NANC) innervation of the rat gastric fundus. ATP (1-30 microM) produced biphasic responses consisting of concentration-dependent relaxations followed by concentration-dependent contractions. Suramin (200 microM) significantly reduced relaxations and abolished contractions to ATP. Under NANC conditions, electrical field stimulation (EFS) induced frequency-dependent relaxations. Suramin (200 microM) and the peptidase alpha-chymotrypsin (1 u ml(-1)) had the same effects on EFS-induced relaxations: their duration was reduced, but their magnitude was unaffected. Cumulative relaxations to vasoactive intestinal peptide (VIP; 0.1-100 nM), and to the VIP analogue pituitary adenylate cyclase activating peptide 1-27 (PACAP; 0.2-100 nM), were almost completely abolished by alpha-chymotrypsin (1 u ml(-1)), and were inhibited by suramin (3-200 microM) in an apparently competitive manner. Schild plot analysis indicated that suramin had pA(2) values of 5.1+/-0.2 (Hill slope=0.9+/-0.2) and 5.6+/-0.1 (Hill slope=1.0+/-0.1), against VIP and PACAP, respectively. Concentration-dependent relaxations to nitric oxide (1-30 microM) and cumulative relaxations to isoprenaline (0.1-300 nM) were not affected by suramin (200 microM). No conclusions can be made regarding the possible involvement of ATP in EFS-induced NANC relaxations. The results suggest that suramin acts as a competitive antagonist at VIP receptors in the rat gastric fundus.

1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl -1, 3-dihydro-2H-benzimidazol-2-one (J-113397) was found to be the first potent nonpeptidyl ORL1 receptor antagonist (K(i): cloned human ORL1=1.8 nM) with high selectivity over other opioid receptors (K(i): 1000 nM for human mu-opioid receptor,10,000 nM for human delta-opioid receptor, and 640 nM for human kappa-opioid receptor). In vitro, J-113397 inhibited nociceptin/orphanin FQ-stimulated [35S]guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) binding to Chinese Hamster Ovary (CHO) cells expressing ORL1 (CHO-ORL1) with an IC(50) value of 5.3 nM but had no effect on [35S]GTP gamma S binding by itself. Schild plot analysis of the [35S]GTP gamma S binding assay and cAMP assay using CHO-ORL1 indicated competitive antagonism of J-113397 on the ORL1 receptor. In CHO cells expressing mu-, delta- or kappa-opioid receptors, J-113397 had no effects on [35S]GTP gamma S binding up to a concentration of 100 nM, indicating selective antagonism of the compound on the ORL1 receptor. In vivo, J-113397, when administered subcutaneously (s.c.), dose-dependently inhibited hyperalgesia elicited by intracerebroventricular (i.c.v.) administration of nociceptin/orphanin FQ in a tail-flick test with mice. An in vitro binding study using mouse brains indicated that J-113397 possesses high affinity for the mouse ORL1 receptor (K(i): 1.1 nM) as well as the human receptor. In summary, J-113397 is the first potent, selective ORL1 receptor antagonist that may be useful in elucidating the physiological roles of nociceptin/orphanin FQ.

Endothelins are a family of potent endogenous mediators that have been implicated in a number of airway and other diseases. Recently, the non-peptide mixed ET(A)/ET(B) endothelin receptor antagonist bosentan has been successfully tested in the treatment of cardiovascular diseases. It was the aim of the present study to characterize the effects of bosentan on the pulmonary actions of endothelin- (ET-1), endothelin-3 (ET-3) and the ET(B)-receptor agonist IRL1620 in the isolated perfused and ventilated rat lung (IPL) and in precision-cut lung slices (PCLS). In the IPL, bosentan completely prevented the IRL1620-induced vasoconstriction (IC50 3 microM). The inhibition by bosentan of ET-1-elicited vasoconstriction showed a biphasic course, reflecting the inhibition of ET(A)-and ET(B)-mediated vasoconstriction (IC50 0.2 microM and 19 microM, respectively). In addition, bosentan prevented the ET-1- (IC50 6 microM) and IRL1620-induced (IC50 3 microM) prostacyclin release. Bosentan also completely prevented the bronchoconstriction induced by IRL1620 in the IPL (IC50 20 microM) and in PCLS (IC50 13 microM). In PCLS, the pD2-values were ET-1 7.20+/-0.23, ET-3 7.51+/-0.27 and IRL1620 7.33+/-0.29. Bosentan at 100 microM caused a rightward shift of the concentration-response curve of ET-1, ET-3 and IRL1620 by a factor of 5, 46 and 64, respectively. In all cases the slope of the Schild regression was lower than unity, disregarding a simple interaction of bosentan with one receptor. With respect to ET-1-induced bronchoconstriction, in the IPL bosentan in concentrations of up to 10 microM aggravated ET-1-induced bronchoconstriction probably due to the blockade of bronchodilatory ET(A)-receptors (IC50 0.3 microM) and even at 100 microM showed only very little protection from ET- -induced bronchoconstriction in the IPL and in the PCLS. The similar IC50-values for ET-1-induced vasoconstriction and bronchodilation suggest that only one type of ET(A)-receptor is involved. The differing IC50-values between IRL1620-induced bronchoconstriction and prostacyclin release, the slope of the Schild regression and the failure of bosentan to prevent the ET-1-induced bronchoconstriction suggest a complex interaction between the known ET-receptors or the existence of unknown ET(B)-receptor subtypes.

1. The antagonist effect of zolertine (4-phenyl-1-[2-(5-tetrazolyl)ethyl]piperazine trihydrochloride), on vascular contraction elicited by noradrenaline in aorta, carotid (alpha1D-adrenoceptors), mesenteric (alpha1A/D-adrenoceptors) and caudal arteries (alpha1A-adrenoceptors) from Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats and rabbit aorta (alpha1B-adrenoceptors), was investigated in endothelium-denuded arterial rings. 2. The selective alpha1D-adrenoceptor agonist, noradrenaline, elicited concentration-dependent contractions in all arterial rings from both species. Noradrenaline selectivity was: carotid = aorta >> mesenteric = rabbit aorta > caudal arteries. 3. The contractile responses induced by noradrenaline were competitively antagonized by zolertine in rat carotid and aorta arteries, yielding pA2 values of WKY, 7.48 +/- 0.18; SHR, 7.43 +/- 0.13 and WKY, 7.57 +/- 0.24; SHR, 7.40 +/- 0.08, respectively. Zolertine was a non-competitive antagonist in some blood vessels as Schild plot slopes were lower than unity. The pKb estimates for zolertine were WKY, 6.98 +/- 0.16; SHR, 6.81 +/- 0.18 in the mesenteric artery, WKY, 5.73 +/- 0.11; SHR, 5.87 +/- 0.25 in the caudal artery and 6.65 +/- 0.09 in rabbit aorta. 4. Competition binding experiments using the alpha1-adrenoceptor antagonist [3H]prazosin showed a zolertine pKi of 6.81 +/- 0.02 in rat liver (alpha1B-adrenoceptors) and 6.35 +/- 0.04 in rabbit liver (alpha1A-adrenoceptors) membranes. 5. Zolertine showed higher affinity for alpha1D-adrenoceptors compared to alpha1A-adrenoceptors, while it had an intermediate affinity for alpha1B-adrenoceptors. The ability of the alpha1-adrenoceptor antagonist zolertine to block alpha1D-adrenoceptor-mediated constriction in different vessels of WKY and SHR rats may explain its antihypertensive efficacy despite its low order of potency.

Characterization of adenosine receptor subtypes was examined on the canine exocrine pancreas using selective adenosine receptor agonists and antagonists in the isolated and blood-perfused pancreas of anaesthetized dogs. Each drug was injected intra-arterially in a single bolus fashion. Graded doses of CGS21680 (1-300 nmol/kg), a selective adenosine A2A receptor agonist, produced dose-dependent increases in the secretory rate of pancreatic juice, with a maximum effect at approximately 30 nmol/kg. However, CPA (1-300 nmol/kg), a selective adenosine A1 receptor agonist, did not cause the pancreatic secretion. CGS21680 (3-30 nmol/kg) and secretin (0.01-0.03 pmol/kg) increased the bicarbonate concentration in pancreatic juice and decreased the protein concentration. CCK-8 (0.1-0.3 pmol/kg) increased the protein concentration but did not alter the bicarbonate concentration. DMPX (5-50 nmol/kg), a weak adenosine A2A receptor antagonist, caused a progressive parallel shift to the right in the dose response curve for CGS21680-induced pancreatic secretion without changes in the maximal response. DPCPX (100 nmol/kg), a selective A1 adenosine receptor antagonist, did not antagonize the CGS21680-induced pancreatic secretion. Schild analysis of the data indicated that the apparent pA2 value for DMPX was 8.3 using CGS21680 as the agonist. The slope of the Schild regression line was not different from 1. When a phosphodiesterase IV inhibitor rolipram (0.1 nmol/kg) was added, pancreatic secretion induced by CGS21680 (10 nmol/kg) and secretin (0.03 pmol/kg) were potentiated, but not that of CCK-8 (0.3 pmol/kg). These results suggest the existence of adenosine A2A receptors in the exocrine cells of the dog pancreas involved in the water and bicarbonate secretory response.

The alpha(1)-adrenoceptor subtype(s) mediating contractions of the rat mesenteric artery were investigated using the agonists methoxamine, cirazoline, P7480 (N-(4-pyridinyl)-1H-indol-1-amine) and subtype-selective antagonists including BMY 7378 (8-(-2(-4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-8-azaspiro(4, 5)decane-7,9,dione dihydrochloride). pA(2) or apparent pK(B) values of antagonists against methoxamine contractions correlated best with its pK(i) values at the cloned alpha(1b)-(0.88), with cirazoline, antagonists affinities correlated equally well with those at alpha(1a)-(0.79) or the alpha(1b)-(0.81) while with P7480 antagonist affinities correlated best with the alpha(1d)-adrenoceptor subtype (0.94). The low affinity estimate for 5-methylurapidil (7.5) against the alpha(1a)-selective cirazoline suggests an alpha(1A)-subtype mediating contraction is unlikely. Shallow Schild plot slopes of subtype selective antagonists against all three agonists are consistent with heterogeneity of alpha(1)-adrenoceptors. P7480 (putative alpha(1D)-adrenoceptor-selective) acts primarily at this subtype and at another which is more likely to be an alpha(1B)- than an alpha(1A)-adrenoceptor. The results with both agonists and antagonists are consistent with contractions of the rat mesenteric artery being mediated via the alpha(1D)- and possibly alpha(1B)-adrenoceptor.

The aim of this study was to determine β-bend structures and the role of the N- and C-terminus in the antagonist hα CGRP8–37 at the rat pulmonary artery CGRP receptor mediating hα CGRP relaxation. Hα CGRP8–37 Pro16 (10−6 M), with a bend-biasing residue (proline) at position 16, did not antagonize hα CGRP responses, while a structure-conserving amino acid (alanine16) at the same position retained antagonist activity (apparent pKB 6.6±0.1; 10−6 M). Hα CGRP8–37 Pro19 (10−6 M), with proline at position 19 was an antagonist (apparent pKB 6.9±0.1). Incorporation of a β-bend forcing residue, BTD (beta-turn dipeptide), at positions 19 and 20 in hα CGRP8–37 (10−6 M) antagonized hα CGRP responses (apparent pKB 7.2±0.2); and BTD at positions 19,20 and 33,34 within hα CGRP8–37 was a competitive antagonist (pA2 7.2; Schild plot slope 1.0±0.1). Hα CGRP8–37 analogues, substituted at the N-terminus by either glycine8 or des-NH2 valine8 or proline8 were all antagonists (apparent pKB 6.9±0.1; (10−6 M), 7.0±0.1 (10−6 M), and pA2 7.0 (slope 1.0±0.2), respectively); while replacements by proline8 together with glutamic acid10,14 in hα CGRP8–37 (10−6 M) or alanine amide37 at the C-terminus of hα CGRP8–37 (10−5 M) were both inactive compounds. In conclusion, possible bioactive structures of hα CGRP8–37 include two β-bends (at 18–21 and 32–35), which were mimicked by BTD incorporation. Within hα CGRP8–37, the N-terminus is not essential for antagonism while the C-terminus may interact directly with CGRP1 receptors in the rat pulmonary artery. Keywords: BTD, β-bend, hα CGRP8–37, hα CGRP, CGRP1 receptor, rat pulmonary artery Introduction Calcitonin gene-related peptide (CGRP) is a 37 residue neuropeptide with numerous biological actions (for review see Poyner et al., 1992; Bell & McDermott, 1996). CGRP receptors have been divided into CGRP1 and CGRP2 (Dennis et al., 1989; 1990; Mimeault et al., 1991; Quirion et al., 1992), mainly based on the antagonist hα CGRP8–37, which has a higher affinity at CGRP1 in the guinea-pig atrium than at the CGRP2 receptor in the rat vas deferens. The lack of pharmacological tools has been a major problem in the further characterization of CGRP receptors. In the vasculature CGRP causes relaxation via endothelium-dependent and independent mechanisms (Marshall, 1992) and different CGRP receptors may be involved (Wisskirchen et al., 1999b). Conformational and modelling studies (Lynch & Kaiser, 1988; Manning, 1989; Hubbard et al., 1991; Breeze et al., 1991; Hakala & Vihinen, 1994) have been undertaken to investigate the structure of CGRP and fragments, and have given evidence of structural features. These include an inducible amphipathic α-helical region between residues 8 to about 18 (Lynch & Kaiser, 1988; Manning, 1989; Breeze et al., 1991; Hubbard et al., 1991), and a tentative assignment to β-bend regions around residues 17 to 21 (Lynch & Kaiser, 1988; Breeze et al., 1991; Hubbard et al., 1991) and 29 to 35 (Hubbard et al., 1991; Hakala & Vihinen, 1994). The amphipathic α-helix has been suggested to be an important feature for interaction of the peptide with its receptors, as its length and sequence is critical for both the biological activity of CGRP (Lynch & Kaiser, 1988) and the antagonist affinity of hα CGRP8–37 (Dennis et al., 1989; Mimeault et al., 1991; 1992; Boulanger et al., 1996). To date, the characterization of this region has been difficult; for instance, in several structure-activity studies the sequential replacement of helical residues with alanine (which conserves structure but removes functionality) has led to equivocal results. With respect to the predicted bend regions, recent structure-activity work suggested the presence of two β-bends in hα CGRP8–37 (Wisskirchen et al., 1999a). The incorporation of BTD (beta-turn dipeptide, which forces a 4 residue β-bend formation; Nagai & Sato 1985) at positions 19,20 and 33,34 identified β-bends at 18–21 and 32–35 as relevant structures for antagonism of hα CGRP8–37 at the CGRP2 receptor in the rat vas deferens. The present study was undertaken to investigate the putative β-bends and the role of the N- and C-terminus of hα CGRP8–37 at the CGRP1 receptor in the rat pulmonary artery (Wisskirchen et al., 1998), by assaying the effect of structurally modified CGRP8–37 analogues against hα CGRP responses. The putative β-bends were examined, using structure-conserving (alanine), bend-biasing (proline) and bend-forcing (BTD) residues incorporated in hα CGRP8–37. The N-terminal region and the C-terminus of hα CGRP8–37 were investigated, using substitutions in position 8 (glycine or proline or des-NH2 valine), in the helical region (proline8 and glutamic acid10,14), and at the C-terminus (alanine amide37).

Muscarinic receptor subtypes controlling the nonselective cationic current in response to carbachol (ICCh) were studied in circular smooth muscle cells of the guinea pig gastric antrum using putative muscarinic agonists and antagonists. Both oxotremorine-M (an M2-selective agonist) and CCh dose-dependently activated the cationic current with EC50 values of 0.21 +/- 0.01 microm and 0.97 +/- 0.06 microM, respectively. In contrast, pilocarpine and McN-A 343 (an M1-selective and a putative M4 agonist) were weak partial agonists. In response to 10/microM CCh, 4-DAMP, methoctramine and pirenzepine dose-dependently inhibited ICCh and had IC50 values of 1.91 +/- 0.2 nM, 0.46 +/- 0.07 microM and 8.33 +/- 0.4 microM, respectively. 4-DAMP, methoctramine and pirenzepine shifted the concentration-response curves of ICCh to the right without significantly reducing the maximal current. Values of the apparent dissociation constant pA2 obtained from Schild plot analysis were 9.24, 7.72 and 6.62 for 4-DAMP, methoctramine and pirenzepine, respectively. Also, pertussis toxin completely blocked ICCh generation. These results suggest that the M2-subtype plays a crucial role in the activation of the ICCh, and a block of the M3-subtype reduces the sensitivity of the M2-mediated response with no significant reduction of maximum response.

The properties of the beta1-/beta2-adrenoceptor partial agonist carteolol were investigated in atypical beta-adrenoceptors on the guinea pig gastric fundus. Carteolol induced concentration-dependent relaxation in this tissue (pD2 = 5.55, intrinsic activity = 0.94). However, a combination of the selective beta1-adrenoceptor antagonist atenolol (100 microM) and the selective beta2-adrenoceptor antagonist butoxamine (100 microM) produced only small rightward shifts in the concentration-response curves of carteolol in the gastric fundus (pD2 = 4.91, intrinsic activity = 0.94). In the presence of both atenolol (100 microM) and butoxamine (100 microM), the non-selective beta1-, beta2- and beta3-adrenoceptor antagonist (+/-)-bupranolol (10-100 microM) caused a concentration-dependent right-ward shift of the concentration-response curves for carteolol in the guinea pig gastric fundus. Schild plot analyses of the effects of (+/-)-bupranolol against carteolol gave the pA2 value of 5.29 and the Schild slope was not significantly different from unity. Furthermore, carteolol (10 microM) weakly but significantly antagonized the relaxant responses to catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline), a selective beta3-adrenoceptor agonist BRL37344 ((R*,R*)-(+/-)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxy-acetic acid sodium salt) and a non-conventional partial beta3-adrenoceptor agonist (+/-)-CGP12177A ([4-[3-[(1,1dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride) in the guinea pig gastric fundus. These results suggest that the partial agonistic effects of carteolol are mediated by atypical beta-adrenoceptors in the guinea pig gastric fundus.

(+/-)-Dobutamine at concentrations < or =10(-5) M did not evoke contractions of rat gastric artery segments. However, when the tissues were contracted with methoxamine, (+/-)-dobutamine evoked concentration-dependent relaxation. The relaxant responses were not significantly affected by propranolol. In the same preparation, propranolol competitively antagonized isoprenaline-induced relaxation with a -log K(B) value of 7.90+/-0.26. (+/-)-Dobutamine did not relax arterial ring segments precontracted with vasopressin (10(-7) M). (+/-)-Dobutamine antagonized noradrenaline-induced contractions of the gastric artery segments. The pA2 value was 6.93+/-0.20, and the slope of the Schild regression line was 1.22+/-0.14. This value (slope) was not significantly different from 1, indicating competitive antagonism. Pretreatment of gastric artery segments with dobutamine before phenoxybenzamine (PBZ) protected against inactivation of alpha1-adrenoceptors by PBZ. The dose ratio of prazosin (3x10(-9) M) and (+/-)-dobutamine (10(-5) M) in combination was close to the expected sum of their individual dose ratios minus 1, indicating interaction with a common site. It was therefore concluded that (+/-)-dobutamine evoked relaxation of rat gastric artery segments by an action not involving beta-adrenoceptor activation but by blocking alpha1-adrenoceptors.

The antagonist activity of a series of diinosine polyphosphates (IpnI, where n=3, 4, 5) was assessed against ATP-activated inward currents at rat P2X1–4 receptors expressed in Xenopus oocytes and studied under voltage-clamp conditions. Diinosine polyphosphates were prepared by the enzymatic degradation of their corresponding diadenosine polyphosphates (e.g., Ap5A into Ip5I) using 5′-adenylic deaminase, and purified using reverse-phase chromatography. Against ATP-responses at rP2X1 receptors, the potency order for antagonism was (pIC50): Ip5I (8.5)>Ip4I (6.3)>Ip3I (>4.5). Ip5I (10–100 nM) caused a concentration-dependent rightwards displacement of the ATP concentration-response curve without reducing the maximum ATP effect. However, the Schild plot was non-linear which indicated Ip5I is not a competitive antagonist. Blockade by micromolar concentrations of Ip5I was not surmountable. Ip4I also behaved as a non-surmountable antagonist. Against ATP-responses at rP2X3 receptors, the potency order for antagonism was (pIC50): Ip4I (6.0)>Ip5I (5.6)>Ip3I (>4.5). Blockade by Ip4I (pA2, 6.75) and Ip5I (pA2, 6.27) was surmountable at micromolar concentrations. Diinosine polyphosphates failed to inhibit ATP-responses at rP2X2 receptors, whereas agonist responses at rP2X4 were reversibly potentiated by Ip4I and Ip5I. None of the parent diadenosine polyphosphates behave as antagonists at rP2X1–4 receptors. Thus, Ip5I acted as a potent and relatively-selective antagonist at the rP2X1 receptor. This dinucleotide pentaphosphate represents a high-affinity antagonist for the P2X1 receptor, at which it acts in a competitive manner at low (100 nM) concentrations but has more complex actions at higher (>100 nM) concentrations. British Journal of Pharmacology (1999) 128, 981–988; doi:10.1038/sj.bjp.0702876

Synthetic peptides (5 to 14 amino acids), identical in sequence to the new amino-terminus of the thrombin receptor generated following cleavage by thrombin, act as thrombin receptor agonist peptides. Whilst thrombin receptor antagonist peptides are known, non-peptide thrombin receptor antagonists have yet to be described. In the present study, we compared the antiplatelet effects of 3-(4-chlorophenyl)-2-(2,4-dichlorobenzoylimino)-5-(methoxycarbonyl methylene)-1,3-thiazolidin-4-one (FR171113), a novel non-peptide thrombin receptor antagonist, with the known thrombin receptor antagonist 3-mercapto-propionyl-Phe-Cha-Cha-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-OH (C186-65), and argatroban, a specific protease inhibitor of thrombin. FR171113 and C186-65 inhibited thrombin-induced platelet aggregation (IC(50)=0.29 microM and 15 microM, respectively) and Ser-Phe-Leu-Leu-Arg-Asn-NH(2) [a synthetic thrombin receptor agonist peptide (TRAP-6)] induced platelet aggregation (0.15 microM and 20 microM, respectively) in human washed platelets. Argatroban potently inhibited thrombin-induced platelet aggregation (IC(50)=3.5 nM), but did not inhibit TRAP-6-induced aggregation even at 100 microM. In contrast, these compounds did not show inhibitory effects on ADP- and collagen-induced aggregation in human platelet-rich plasma even at 100 microM. FR171113 caused a parallel shift to the right of the concentration-response curve describing aggregation induced by TRAP-6. The Schild plot of the data had a slope of -0.840 (r=0.98) and the pA(2) was 7.29. In protease activity studies using a chromogenic substrate, argatroban inhibited thrombin protease activity in a dose-dependent manner, whereas FR171113 and C186-65 were inactive, even at 100 microM. Additionally, only argatroban displayed dose-dependent prolongation of thrombin time, activated partial thromboplastin time and prothrombin time. FR171113 and C186-65 showed no effects, even at a concentration of 100 microM. These results suggest that FR171113 has a similar mode of action to C186-65, but with more potent antiplatelet activity. In conclusion, FR171113 is suggested to be the first example of a non-peptide thrombin receptor antagonist.

We previously suggested that the existence of atypical beta/beta3-adrenoceptor with pA2-values for bupranolol, a non-selective beta-adrenoceptor antagonist, against BRL37344 and noradrenaline were 5.79 and 5.53 in guinea pig taenia caecum, respectively. We furthermore determined the affinity of bupranolol to subclassify atypical beta/beta3-adrenoceptor in rat oesophageal muscularis mucosae, because it is rich in atypical beta/beta3-adrenoceptor. BRL37344 and noradrenaline produced a concentration-dependent relaxation of rat oesophageal muscularis mucosae. The responses to BRL37344 and noradrenaline were resistant to 3x10(-6) M propranolol, 10(-4) M atenolol, and 10(-4) M butoxamine. However, bupranolol antagonized the responses to BRL37344 and noradrenaline in a concentration-dependent manner. Schild plot analyses of bupranolol against BRL37344 and noradrenaline gave pA2-values of 7.06 and 6.96, respectively. These results suggest that bupranolol can distinguish the difference in affinity between atypical beta/beta3-adrenoceptors in rat oesophageal muscularis mucosae and guinea pig taenia caecum. The difference in behavior of bupranolol confirms the existence of some atypical beta/beta3-adrenoceptors subtypes.