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1.
- In segments of human right atrial appendages and pulmonary arteries preincubated with [3H]-noradrenaline and superfused with physiological salt solution containing desipramine and corticosterone, the involvement of imidazoline receptors in the modulation of [3H]-noradrenaline release was investigated.
- In human atrial appendages, the guanidines aganodine and DTG (1,3-di(2-tolyl)guanidine) which activate presynaptic imidazoline receptors, inhibited electrically-evoked [3H]-noradrenaline release. The inhibition was not affected by blockade of α2-adrenoceptors with 1 μM rauwolscine, but antagonized by extremely high concentrations of this drug (10 and/or 30 μM; apparent pA2 against aganodine and DTG: 5.55 and 5.21, respectively).
- In the presence of 1 μM rauwolscine, [3H]-noradrenaline release in human atrial appendages was also inhibited by the imidazolines idazoxan and cirazoline, but not by agmatine and noradrenaline. The inhibitory effects of 100 μM idazoxan and 30 μM cirazoline were abolished by 30 μM rauwolscine.
- In the atrial appendages, the rank order of potency of all guanidines and imidazolines for their inhibitory effect on electrically-evoked [3H]-noradrenaline release in the presence of 1 μM rauwolscine was: aganodine⩾BDF 6143 [4-chloro-2-(2-imidazolin-2-yl-amino)-isoindoline]>DTG⩾clonidine>cirazoline>idazoxan (BDF 6143 and clonidine were previously studied under identical conditions). This potency order corresponded to that previously determined at the presynaptic imidazoline receptors in the rabbit aorta.
- When, in the experiments in the human pulmonary artery, rauwolscine was absent from the superfusion fluid, the concentration-response curve for BDF 6143 (a mixed α2-adrenoceptor antagonist/imidazoline receptor agonist) for its facilitatory effect on electrically-evoked [3H]-noradrenaline release was bell-shaped. In the presence of 1 μM rauwolscine, BDF 6143 and cirazoline concentration-dependently inhibited the evoked [3H]-noradrenaline release.
- In human atrial appendages, non-adrenoceptor [3H]-idazoxan binding sites were identified and characterized. The binding of [3H]-idazoxan was specific, reversible, saturable and of high affinity (KD: 25.5 nM). The specific binding of [3H]-idazoxan (defined by cirazoline 0.1 mM) to membranes of human atrial appendages was concentration-dependently inhibited by several imidazolines and guanidines, but not by rauwolscine and agmatine. In most cases, the competition curves were best fitted to a two-site model.
- The rank order of affinity for the high affinity site (in a few cases for the only detectable site; cirazoline=idazoxan>BDF 6143>DTG⩾clonidine) is compatible with the pharmacological properties of I2-imidazoline binding sites, but is clearly different from the rank order of potency for inhibiting evoked noradrenaline release from sympathetic nerves in the same tissue.
- It is concluded that noradrenaline release in the human atrium and, less well established, in the pulmonary artery is inhibited via presynaptic imidazoline receptors. These presynaptic imidazoline receptors appear to be related to those previously characterized in rabbit aorta and pulmonary artery, but differ clearly from I1 and I2 imidazoline binding sites.
2.
- Interations were investigated between loreclezole, chlormethiazole and pentobarbitone as potentiators of depolarization responses mediated by γ-aminobutyric acidA (GABAA) receptors on afferent nerve terminals in the rat cuneate nucleus in vitro. These drugs were also compared as modulators of [3H]-flunitrazepam (FNZ) binding to synaptic membranes prepared from rat whole brain homogenate.
- In rat cuneate nucleus slices, the drugs shifted muscimol log dose–response lines to the left in an approximately parallel fashion with the result that 200 μM chlormethiazole potentiated muscimol responses by 0.567±0.037 log unit (mean±s.e.mean, n=4) while loreclezole gave a maximal potentiation at 10 μM of only 0.121±0.037 (n=6) log unit and 0.071±0.039 (n=22) at 50 μM.
- While 50 μM chlormethiazole and 30 μM pentobarbitone showed no significant interactions between each other when potentiating muscimol responses in combination, 50 μM loreclezole in combination with either chlormethiazole or pentobarbitone attenuated their potentiating effects, possibly by inducing desensitization of GABAA receptors.
- In the [3H]-FNZ binding studies on well-washed membranes, loreclezole enhanced binding to a maximum of 47.3±2.83% of control (mean±s.e.mean, n=3) at 300 μM. Scatchard analysis revealed no change in Bmax but a decrease in KD for [3H]-FNZ from 3.9±0.29 nM to 2.7±0.10 nM (mean±s.e.mean, n=4) in the presence of 100 μM loreclezole. In contrast, 100 μM chlormethiazole caused no potentiation. A small component of the enhancement by loreclezole could be blocked by 100 μM bicuculline and could also be blocked by 100 μM chlormethiazole. It seems likely that the effects on [3H]-FNZ binding are due predominantly to direct actions of the drugs on the GABAA receptor and are separate from the GABA-potentiating effects.
- The results indicate distinctly different profiles of action for loreclezole, chlormethiazole and pentobarbitone on GABAA receptors.
3.
Jayne Cartmell Geo Adam Sylvie Chaboz Robert Henningsen John A Kemp Agnes Klingelschmidt Veit Metzler Frederick Monsma Hervé Schaffhauser Jürgen Wichmann Vincent Mutel 《British journal of pharmacology》1998,123(3):497-504
- The binding of the new selective group II metabotropic glutamate receptor radioligand, [3H]-(2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine ([3H]-DCG IV), was characterized in rat mGlu2 receptor-transfected CHO cell membranes.
- [3H]-DCG IV binding was pH-dependent, but was not sensitive to temperature. Saturation analysis showed the presence of a single binding site, with a Kd value of 160 nM and a Bmax value of 10 pmol mg−1 protein. Binding was not sensitive to Na+-dependent glutamate uptake blockers or Cl−-dependent glutamate binding inhibitors. Furthermore, up to concentrations of 1 mM, the glutamate ionotropic receptor agonists, N-methyl-D-aspartic acid (NMDA), (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate, did not affect [3H]-DCG IV binding.
- Of the compounds observed to inhibit [3H]-DCG IV binding, the most potent were the recently described selective group II agonist, (+)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylate (LY 354740; Ki value 16 nM) and antagonist, 2-amino-2-(2-carboxycyclopropan-1-yl)-3-(dibenzopyran-4-yl) propanoic acid (LY 341495; Ki value 19 nM). As expected, for a G-protein-coupled receptor, guanosine-5′-O-(3-thiotriphosphate) (GTPγS) inhibited [3H]-DCG IV binding in a concentration-dependent manner, with an IC50 value of 12 nM.
- A highly significant correlation was observed between the potencies of compounds able to inhibit [3H]-DCG IV binding and potencies obtained for agonist activity in a GTPγ35S binding functional assay. In addition, these studies identified a number of compounds with previously unknown activity at mGlu2 receptors, including L(+)-2-amino-3-phosphonopropionic acid (L-AP3), L(+)-2-amino-5-phosphonopentanoic acid (L-AP5), 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (R-CPP), N-acetyl-L-aspartyl-L-glutamic acid (NAAG) and (RS)-α-methylserine-O-phosphate (MSOP).
4.
D Smart R A Hirst K Hirota D K Grandy D G Lambert 《British journal of pharmacology》1997,120(6):1165-1171
- The rat μ-opioid receptor has recently been cloned, yet its second messenger coupling remains unclear. The endogenous μ-opioid receptor in SH-SY5Y cells couples to phospholipase C (PLC), increases [Ca2+]i and inhibits adenylyl cyclase (AC). We have examined the effects of μ-opioid agonists on inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), [Ca2+]i and adenosine 3′ : 5′-cyclic monophosphate (cyclic AMP) formation in Chinese hamster ovarian (CHO) cells transfected with the cloned μ-opioid receptor.
- Opioid receptor binding was assessed with [3H]-diprenorphine ([3H]-DPN) as a radiolabel. Ins(1,4,5)P3 and cyclic AMP were measured by specific radioreceptor assays. [Ca2+]i was measured fluorimetrically with Fura-2.
- Scatchard analysis of [3H]-DPN binding revealed that the Bmax varied between passages. Fentanyl (10 pM–1 μM) dose-dependently displaced [3H]-DPN, yielding a curve which had a Hill slope of less than unity (0.6±0.1), and was best fit to a two site model, with pKi values (% of sites) of 9.97±0.4 (27±4.8%) and 7.68±0.07 (73±4.8%). In the presence of GppNHp (100 μM) and Na+ (100 mM), the curve was shifted to the right and became steeper (Hill slope=0.9±0.1) with a pKi value of 6.76±0.04.
- Fentanyl (0.1 nM–1 μM) had no effect on basal, but dose-dependently inhibited forskolin (1 μM)-stimulated, cyclic AMP formation (pIC50=7.42±0.23), in a pertussis toxin (PTX; 100 ng ml−1 for 24 h)-sensitive and naloxone-reversible manner (Ki=1.7 nM). Morphine (1 μM) and [D-Ala2, MePhe4, gly(ol)5]-enkephalin (DAMGO, 1 μM) also inhibited forskolin (1 μM)-stimulated cyclic AMP formation, whilst [D-Pen2, D-Pen5], enkephalin (DPDPE, 1 μM) did not.
- Fentanyl (0.1 nM–10 μM) caused a naloxone (1 μM)-reversible, dose-dependent stimulation of Ins(1,4,5)P3 formation, with a pEC50 of 7.95±0.15 (n=5). PTX (100 ng ml−1 for 24 h) abolished, whilst Ni2+ (2.5 mM) inhibited (by 52%), the fentanyl-induced Ins(1,4,5)P3 response. Morphine (1 μM) and DAMGO (1 μM), but not DPDPE (1 μM), also stimulated Ins(1,4,5)P3 formation. Fentanyl (1 μM) also caused an increase in [Ca2+]i (80±16.4 nM, n=6), reaching a maximum at 26.8±2.5 s. The increase in [Ca2+]i remained elevated until sampling ended (200 s) and was essentially abolished by the addition of naloxone (1 μM). Pre-incubation with naloxone (1 μM, 3 min) completely abolished fentanyl-induced increases in [Ca2+]i.
- In conclusion, the cloned μ-opioid receptor when expressed in CHO cells stimulates PLC and inhibits AC, both effects being mediated by a PTX-sensitive G-protein. In addition, the receptor couples to an increase in [Ca2+]i. These findings are consistent with the previously described effector-second messenger coupling of the endogenous μ-opioid receptor.
5.
- Clozapine has recently been claimed to behave as a selective and full agonist at the cloned m4 muscarinic receptor artificially expressed in Chinese hamster ovary (CHO) cells. In the present study we have investigated whether clozapine could activate the rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity, considered as pharmacologically equivalent to the m4 gene product. In addition, we have examined the effect of the drug on various functional responses following the activation of the cloned m4 receptor expressed in CHO cells.
- In rat striatum, clozapine (1 nM–10 μM) caused a slight inhibition of forskolin-stimulated adenylyl cyclase activity, which was not counteracted by 10 μM atropine. On the other hand, clozapine antagonized the inhibitory effect of acetylcholine with a pA2 value of 7.51. Moreover, clozapine (1 μM) failed to inhibit dopamine D1 receptor stimulation of adenylyl cyclase activity, but counteracted the inhibitory effect of carbachol (CCh). Clozapine displaced [3H]-N-methylscopolamine ([3H]-NMS) bound to striatal M4 receptors with a monophasic inhibitory curve and a pKi value of 7.69. The clozapine inhibition was not affected by the addition of guanosine-5′-O-(thio)triphosphate (GTPγS).
- In intact CHO cells, clozapine inhibited forskolin-stimulated cyclic AMP accumulation with an EC50 of 31 nM. This effect was antagonized by atropine. CCh produced a biphasic effect on cyclic AMP levels, inhibiting at concentrations up to 1 μM (EC50=50 nM) and stimulating at higher concentrations (EC50=7 μM). Clozapine (0.3–5 μM) antagonized the CCh stimulation of cyclic AMP with a pKi value of 7.47. Similar results were obtained when the adenylyl cyclase activity was assayed in CHO cell membranes.
- In CHO cells pretreated with the receptor alkylating agent 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (10 μM), the maximal inhibitory effect of clozapine on cyclic AMP formation was markedly reduced, whereas the CCh inhibitory curve was shifted to the right with no change in the maximum.
- As in rat striatum, in CHO cell membranes the displacement of [3H]-NMS binding by clozapine yielded a monophasic curve which was not affected by GTPγS.
- Clozapine (10 nM–10 μM) had a small stimulant effect (∼20%) on the binding of [35S]-GTPγS to CHO cell membranes, whereas CCh caused a 250% increase of radioligand binding. Moreover, clozapine (50 nM–5 μM) antagonized the CCh-stimulated [35S]-GTPγS binding with a pA2 value of 7.48.
- These results show that at the striatal M4 receptors clozapine is a potent and competitive antagonist, whereas at the cloned m4 receptor it elicits both agonist and antagonist effects. Thus, clozapine behaves as a partial agonist, rather than as a full agonist, at the m4 receptor subtype, with intrinsic activity changing as a function of the coupling efficiency of the receptor to effector molecules.
6.
Annette Hambrock Cornelia L?ffler-Walz Yoshihisa Kurachi Ulrich Quast 《British journal of pharmacology》1998,125(3):577-583
- The binding of modulators of the ATP-sensitive K+ channel (KATP channel) to the murine sulphonylurea receptor, SUR2B, was investigated. SUR2B, a proposed subunit of the vascular KATP channel, was expressed in HEK 293 cells and binding assays were performed in membranes at 37°C using the tritiated KATP channel opener, [3H]-P1075.
- Binding of [3H]-P1075 required the presence of Mg2+ and ATP. MgATP activated binding with EC50 values of 10 and 3 μM at free Mg2+ concentrations of 3 μM and 1 mM, respectively. At 1 mM Mg2+, binding was lower than at 3 μM Mg2+.
- [3H]-P1075 saturation binding experiments, performed at 3 mM ATP and free Mg2+ concentrations of 3 μM and 1 mM, gave KD values of 1.8 and 3.4 nM and BMAX values of 876 and 698 fmol mg−1, respectively.
- In competition experiments, openers inhibited [3H]-P1075 binding with potencies similar to those determined in rings of rat aorta.
- Glibenclamide inhibited [3H]-P1075 binding with Ki values of 0.35 and 2.4 μM at 3 μM and 1 mM free Mg2+, respectively. Glibenclamide enhanced the dissociation of the [3H]-P1075-SUR2B complex suggesting a negative allosteric coupling between the binding sites for P1075 and the sulphonylureas.
- It is concluded that an MgATP site on SUR2B with μM affinity must be occupied to allow opener binding whereas Mg2+ concentrations ⩾10 μM decrease the affinities for openers and glibenclamide. The properties of the [3H]-P1075 site strongly suggest that SUR2B represents the drug receptor of the openers in vascular smooth muscle.
7.
- In vitro receptor autoradiography using [3H]-L-2-amino-4-phosphonobutyrate ([3H]-L-AP4) binding to sections of rat brain has been characterized and shown to most likely represent labelling of group III metabotropic glutamate receptors.
- Specific [3H]-L-AP4 binding to rat brain sections was observed at high densities in the molecular layer of the cerebellar cortex and the outer layer of the superior colliculus. Moderate levels were observed throughout the cerebral cortex, in the molecular layer of the hippocampal dentate gyrus, in thalamus, striatum, substantia nigra and in the medial geniculate nucleus. Low levels of [3H]-L-AP4 binding were found in other regions of the hippocampal formation, in the entorhinal cortex and the granule cell layer of cerebellum.
- Inhibitors of sodium- or calcium/chloride-dependent glutamate uptake did not displace [3H]-L-AP4 binding to rat brain sections indicating that the observed binding does not represent [3H]-L-AP4 uptake via these carriers. Furthermore, in contrast to [3H]-L-AP4 uptake into cerebellar membranes, [3H]-L-AP4 binding to brain sections was sensitive to guanosine-5′-O-(3-thio)trisphosphate-γ-S.
- In the molecular layer of the cerebellar cortex, [3H]-L-AP4 binding showed a maximal binding density (Bmax) of 0.52±0.06 pmol mg−1 tissue and an affinity (Kd) of 346 nM. The rank order of affinity for displacement of [3H]-L-AP4 binding to rat brain sections was: L-AP4>L-serine-O-phosphate>glutamate>(L)-2-aminomethyl-4-phosphonobutanoate>(1S,3R)-1-aminocyclopentane-1,3-dicarboxylate which is in agreement with a group III metabotropic glutamate receptor pharmacology.
8.
Kimihito Tashima Roman Korolkiewicz Masafumi Kubomi Koji Takeuchi 《British journal of pharmacology》1998,123(7):1395-1402
- The binding of [3H]-P1075, a potent opener of adenosine-5′-triphosphate-(ATP)-sensitive K+ channels, was studied in a crude heart membrane preparation of the rat, at 37°C.
- Binding required MgATP. In the presence of an ATP-regenerating system, MgATP supported [3H]-P1075 binding with an EC50 value of 100 μM and a Hill coefficient of 1.4.
- In saturation experiments [3H]-P1075 binding was homogeneous with a KD value of 6±1 nM and a binding capacity (Bmax) of 33±3 fmol mg−1 protein.
- Upon addition of an excess of unlabelled P1075, the [3H]-P1075-receptor complex dissociated in a mono-exponential manner with a dissociation rate constant of 0.13±0.01 min−1. If a bi-molecular association mechanism was assumed, the dependence of the association kinetics on label concentration gave an association rate constant of 0.030±0.003 nM−1 min−1. From the kinetic experiments the KD value was calculated as 4.7±0.6 nM.
- Openers of the ATP-sensitive K+ channel belonging to different structural classes inhibited specific [3H]-P1075 binding in a monophasic manner to completion; an exception was minoxidil sulphate where maximum inhibition was 68%. The potencies of the openers in this assay agree with published values obtained in rat cardiocytes and are on average 3.5 times lower than those determined in rat aorta.
- Sulphonylureas, such as glibenclamide and glibornuride and the sulphonylurea-related carboxylate, AZ-DF 265, inhibited [3H]-P1075 binding with biphasic inhibition curves. The high affinity component comprised about 60% of the curves with the IC50 value of glibenclamide being ≈amp;90 nM; affinities for the low affinity component were in the μM concentration range. The fluorescein derivative, phloxine B, showed a monophasic inhibition curve with an IC50 value of 6 μM, a maximum inhibition of 94% and a Hill coefficient of 1.5.
- It is concluded that binding studies with [3H]-P1075 are feasible in rat heart membranes in the presence of MgATP and of an ATP-regenerating system. The pharmacological profile of the [3H]-P1075 binding sites in the cardiac preparation, which probably contains sulphonylurea receptors (SURs) from cardiac myocytes (SUR2A) and vascular smooth muscle cells (SUR2B), differs from that expected for SUR2A and SUR2B.
9.
10.
- The tachykinin receptors mediating contraction of isolated longitudinal strips of the guinea-pig oesophageal body were characterized with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) as well as the analogues, [Sar9,Met(O2)11]SP, [Nle10]NKA(4–10) and [MePhe7]NKB, selective for NK1, NK2 and NK3, receptors, respectively. Experiments were performed both in the absence and presence of a cocktail of peptidase inhibitors, captopril (1 μM), thiorphan (1 μM) and amastatin (20 μM), in order to determine whether membrane bound proteases are important in the metabolism of tachykinins in this preparation.
- All agonists produced concentration-dependent contractile effects. The presence of the peptidase inhibitors shifted the concentration-response curves of SP, [Nle10]NKA(4–10) and [MePhe7]NKB significantly leftwards and the concentration-response curve of NKB was shifted significantly rightwards. However, the EC50 values were significantly different only for [Nle10]NKA(4–10) and NKB.
- In the presence of the peptidase inhibitors, the EC50 values of the selective agonists, [MePhe7]NKB (0.6 nM) and [Nle10]NKA(4–10) (66 nM) indicated the presence of both tachykinin NK3 and NK2 receptors. [MePhe7]NKB produced less than 50% of the maximal response obtained with the other agonists. Since [Sar9,Met(O2)11]SP produced a small response in the nanomolar concentration range in about 30% of the preparations tested, it is possible that some NK1 receptors were also present.
- Assuming competitive antagonism, the NK2-selective antagonist SR 48,968 (30 nM) gave apparent pKB values of 8.13 and 8.65 for [Nle10]NKA(4–10) in the absence and presence of peptidase inhibitors, respectively, supporting the presence of NK2 receptors.
- The NK3-selective antagonist SR 142,801 (0.1 μM), suppressed responses to low (0.1–10 nM) concentrations of [MePhe7]NKB. These contractile responses to [MePhe7]NKB were also abolished by atropine (0.6 μM) suggesting that this response was mediated via cholinergic nerves.
- It is concluded that the guinea-pig oesophagus is a complex system which has both NK2 and NK3 receptors and possibly some NK1 receptors as well.
11.
James J Rauh Eric Benner Michael E Schnee Daniel Cordova Caleb W Holyoke Michael H Howard Donglin Bai Steven D Buckingham Michael L Hutton Alain Hamon Richard T Roush David B Sattelle 《British journal of pharmacology》1997,121(7):1496-1505
- The radiolabelled bicyclic dinitrile, [3H]-3,3-bis-trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([3H]-BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undecimpunctata howardi) and other insects. A variety of γ-aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachlor (IC50, 35±3 nM) and dieldrin (IC50, 93±7 nM), displaced [3H]-BIDN from rootworm membranes. When tested at 100 μM, 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane(EBOB), 4-t-butyl-2,6,7-trioxa-1-phosphabicyclo[2.2.2]octane-1-thione (TBPS), 1-phenyl-4-t-butyl-2,6,7-trioxabicyclo[2.2.2]octane (TBOB) and picrotoxin failed to displace 50% of [3H]-BIDN binding to rootworm membranes indicating that the bicyclic dinitrile radioligand probes a site distinct from those identified by other convulsant radioligands.
- Dissociation studies showed that dieldrin, ketoendrin, toxaphene, heptachlor epoxide and α and β endosulphan displace bound [3H]-BIDN from rootworm membranes by a competitive mechanism.
- Rat brain membranes were also shown to possess a population of saturable, specific [3H]-BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the insecticidal GABAergic convulsants that displaced [3H]-BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more effective in rootworm.
- Functional GABA-gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABAB receptors were unaffected.
- Expression in Xenopus oocytes of either rat brain mRNA, or cDNA-derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo-oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA-gated Cl− channels to which a number of insecticidally-active molecules bind.
12.
- Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca2+ ([Ca2+]i), phosphoinositide (PI) turnover, and arachidonic acid (AA) release, the causal relationship between these signalling pathways is still unclear. In the present study, we investigated the involvement of phosphoinositide-dependent phospholipase C (PI-PLC) activation, Ca2+ increase and protein kinase activation in UTP-induced AA release. The effects of stimulating RAW 264.7 cells with thapsigargin, which cannot activate the inositol phosphate (IP) cascade, but results in the release of sequestered Ca2+ and an influx of extracellular Ca2+, was compared with the effects of UTP stimulation to elucidate the multiple regulatory pathways for cPLA2 activation.
- In RAW 264.7 cells UTP (100 μM) and thapsigargin (1 μM) caused 2 and 1.2 fold increases, respectively, in [3H]-AA release. The release of [3H]-AA following treatment with UTP and thapsigargin were non-additive, totally abolished in the Ca2+-free buffer, BAPTA (30 μM)-containing buffer or in the presence of the cPLA2 inhibitor MAFP (50 μM), and inhibited by pretreatment of cells with pertussis toxin (100 ng ml−1) or 4-bromophenacyl bromide (100 μM). By contrast, aristolochic acid (an inhibitor of sPLA2) had no effect on UTP and thapsigargin responses.
- (10 μ U73122M) and neomycin (3 mM), inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83% inhibition, respectively) and AA release (76% and 58%, respectively), accompanied by a decrease in the [Ca2+]i rise.
- Wortmannin attenuated the IP response of UTP in a concentration-dependent manner (over the range 10 nM–3 μM), and reduced the UTP-induced AA release in parallel. RHC 80267 (30 μM), a specific diacylglycerol lipase inhibitor, had no effect on UTP-induced AA release.
- Short-term treatment with PMA (1 μM) inhibited the UTP-stimulated accumulation of IP and increase in [Ca2+]i, but had no effect on the release of AA. In contrast, the AA release caused by thapsigargin was increased by PMA.
- The role of PKC in UTP- and thapsigargin-mediated AA release was shown by the blockade of these effects by staurosporine (1 μM), Ro 31-8220 (10 μM), Go 6976 (1 μM) and the down-regulation of PKC.
- Following treatment of cells with SK&F 96365 (30 μM), thapsigargin-, but not UTP-, induced Ca2+ influx, and the accompanying AA release, were down-regulated.
- Neither PD 98059 (100 μM), MEK a inhibitor, nor genistein (100 μM), a tyrosine kinase inhibitor, had any effect on the AA responses induced by UTP and thapsigargin.
- We conclude that UTP-induced cPLA2 activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca2+, which is essential for the activation of cPLA2 by UTP and thapsigargin. The [Ca2+]i-dependent AA release that follows treatment with both stimuli was potentiated by the activity of protein kinase C (PKC). A pertussis toxin-sensitive pathway downstream of the increase in [Ca2+]i was also shown to be involved in AA release.
13.
Lucia Spicuzza Mark A Giembycz Peter J Barnes Maria G Belvisi 《British journal of pharmacology》1998,123(6):1246-1252
- We have demonstrated recently that exogenous prostaglandin E2 (PGE2) inhibits electrical field stimulation (EFS)-induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea-pig trachea. In the present study, we have attempted to characterize the pre-junctional prostanoid receptor(s) responsible for the inhibitory action of PGE2 and to assess whether other prostanoids modulate, at a prejunctional level, cholinergic neurotransmission in guinea-pig trachea. To this end, we have investigated the effect of a range of both natural and synthetic prostanoid agonists and antagonists on EFS-evoked [3H]-ACh release.
- In epithelium-denuded tracheal strips pretreated with indomethacin (10 μM), PGE2 (0.1 nM–1 μM) inhibited EFS-evoked [3H]-ACh release in a concentration-dependent manner with an EC50 and maximal effect of 7.62 nM and 74% inhibition, respectively. Cicaprost, an IP-receptor agonist, PGF2α and the stable thromboxane mimetic, U46619 (each at 1 μM), also inhibited [3H]-ACh release by 48%, 41% and 35%, respectively. PGD2 (1 μM) had no significant effect on [3H]-ACh release.
- The selective TP-receptor antagonist, ICI 192,605 (0.1 μM), completely reversed the inhibition of cholinergic neurotransmission induced by U-46619, but had no significant effect on similar responses effected by PGE2 and PGF2α.
- A number of EP-receptor agonists mimicked the ability of PGE2 to inhibit [3H]-ACh release with a rank order of potency: GR63799X (EP3-selective)>PGE2>M&B 28,767 (EP3 selective)>17-phenyl-ω-trinor PGE2 (EP1-selective). The EP2-selective agonist, AH 13205 (1 μM), did not affect EFS-induced [3H]-ACh release.
- AH6809 (10 μM), at a concentration 10 to 100 times greater than its pA2 at DP-, EP1- and EP2-receptors, failed to reverse the inhibitory effect of PGE2 or 17-phenyl-ω-trinor PGE2 on [3H]-ACh release.
- These results suggest that PGE2 inhibits [3H]-ACh release from parasympathetic nerves supplying guinea-pig trachea via an interaction with prejunctional prostanoid receptors of the EP3-receptor subtype. Evidence for inhibitory prejunctional TP- and, possibly, IP-receptors was also obtained although these receptors may play only a minor role in suppressing [3H]-ACh release when compared to receptors of the EP3-subtype. However, the relative importance of the different receptors will depend not only on the sensitivity of guinea-pig trachea to prostanoids but on the nature of the endogenous ligands released locally that have activity on parasympathetic nerves.
14.
Liang Li Kirsi Vaali Ilari Paakkari Heikki Vapaatalo 《British journal of pharmacology》1998,123(7):1337-1342
- The aim of the present study was to investigate the effects of bradykinin and [des-Arg9]-bradykinin and their relaxant mechanisms in the mouse isolated trachea.
- In the resting tracheal preparations with intact epithelium, bradykinin and [des-Arg9]-bradykinin (each drug, 0.01–10 μM) induced neither contraction nor relaxation. In contrast, bradykinin (0.01–10 μM) induced concentration-dependent relaxation when the tracheal preparations were precontracted with methacholine (1 μM). The relaxation induced by bradykinin was inhibited by the B2 receptor antagonist, D-Arg0-[Hyp3,Thi5,D-Tic7,Oic8]-bradykinin (Hoe 140, 0.01–1 μM) in a concentration-dependent manner whereas the B1 receptor antagonist, [des-Arg9,Leu8]-bradykinin (0.01–1 μM), had no inhibitory effect on bradykinin-induced relaxation. [des-Arg9]-bradykinin (0.01–10 μM) also caused concentration-dependent relaxation after precontraction with methacholine. The relaxation induced by [des-Arg9]-bradykinin was concentration-dependently inhibited by the B1 receptor antagonist, [des-Arg9,Leu8]-bradykinin (0.01–1 μM), whereas the B2 receptor antagonist, Hoe 140 (0.01–1 μM) was without effect.
- In the presence of the cyclo-oxygenase inhibitor, indomethacin (0.01–1 μM), the relaxations induced by bradykinin and [des-Arg9]-bradykinin were inhibited concentration-dependently.
- Two nitric oxide (NO) biosynthesis inhibitors NG-nitro-L-arginine methyl ester (L-NAME, 100 μM) and NG-nitro-L-arginine (L-NOARG, 100 μM) had no inhibitory effects on the relaxations induced by bradykinin and [des-Arg9]-bradykinin. Neither did the selective inhibitor of the soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 μM) inhibit the relaxations induced by bradykinin and [des-Arg9]-bradykinin.
- Prostaglandin E2 (PGE2, 0.01–33 μM) caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. Indomethacin (1 μM) and ODQ (10 μM) exerted no inhibitory effects on the relaxation induced by PGE2.
- The NO-donor, sodium nitroprusside (SNP; 0.01–100 μM) also caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. ODQ (0.1–1 μM) concentration-dependently inhibited the relaxation induced by SNP.
- These data demonstrate that bradykinin and [des-Arg9]-bradykinin relax the mouse trachea precontracted with methacholine by the activation of bradykinin B2-receptors and B1-receptors, respectively. The stimulation of bradykinin receptors induces activation of the cyclo-oxygenase pathway, leading to the production of relaxing prostaglandins. The NO pathway is not involved in the bradykinin-induced relaxation. The relaxation caused by NO-donors in the mouse trachea is likely to be mediated via activation of soluble guanylate cyclase.
15.
Elizabeth C Akam Alan M Carruthers Stefan R Nahorski R A John Challiss 《British journal of pharmacology》1997,121(6):1203-1209
- The activation of G proteins by type 1α metabotropic glutamate receptors (mGluRs) in membranes from recombinant baby hamster kidney cells expressing the cloned rat mGluR1α receptor has been studied by use of a [35S]-guanosine 5′-[γ-thio]triphosphate ([35S]-GTPγS) binding assay.
- L-Glutamate increased the rate of [35S]-GTPγS binding in a concentration-dependent manner (−logEC50 (M) 5.25±0.07), with an optimal (62.4±1.6%) increase over basal binding being observed following 60 min incubation at 30°C with 70 pM [35S]-GTPγS, 1 μM GDP, 10 mM MgCl2, 100 mM NaCl and 100 μg membrane protein ml−1. The L-glutamate (100 μM)-stimulated increase in [35S]-GTPγS binding was totally prevented in the presence of the group I mGluR antagonist (S)-4-carboxy-3-hydroxyphenylglycine (300 μM).
- Quantitative analysis of the affinity and number of G proteins activated by a maximally effective concentration of L-glutamate revealed an equilibrium dissociation constant (KD) for [35S]-GTPγS binding of 0.76±0.20 nM and a maximal number of GTPγS-liganded G proteins (Bmax) of 361±30 fmol mg−1 protein.
- Metabotropic glutamate receptor agonists, quisqualate (−logEC50 (M) 6.74±0.06), 1S,3R-ACPD (4.64±0.08) and (S)-3,5-dihydroxyphenylglycine (5.16±0.23) also increased [35S]-GTPγS binding in a concentration-dependent manner, with the latter two agents behaving as partial agonists.
- (+)-α-Methylcarboxyphenylglycine (300 μM) caused a parallel rightward shift of the L-glutamate concentration-effect curve for [35S]-GTPγS binding, allowing an antagonist equilibrium dissociation constant (KD) of 34.0±7.8 μM to be calculated for this mGluR antagonist.
- Pretreatment of BHK-mGluR1α cells with a concentration of pertussis toxin (PTX) shown to be maximally effective (100 ng ml−1, 24 h) before membrane preparation resulted in a marked decrease in agonist-stimulated [35S]-GTPγS binding (by 66.0±0.9%), and an altered concentration-effect relationship for agonist-stimulated [35S]-GTPγS binding by the residual PTX-insensitive G-protein population.
- The modulation of [35S]-GTPγS binding by agonists and antagonists in membranes from recombinant cells provides an excellent system in which to study mGluR interactions with PTX-sensitive and -insensitive G proteins.
16.
Emmanuel Hermans Muriel Geurts Jean-Marie Maloteaux 《British journal of pharmacology》1997,121(8):1817-1823
- The functional interaction of the cloned rat neurotensin receptor with intracellular G-proteins was investigated by studying the binding of the radiolabelled guanylyl nucleotide analogue [35S]-GTPγS induced by neurotensin to membranes prepared from transfected Chinese hamster ovary (CHO) cells.
- The agonist-induced binding of [35S]-GTPγS was only detected in the presence of NaCl in the incubation buffer. However, it was also demonstrated that the binding of [3H]-neurotensin to its receptor was inhibited by NaCl. In the presence of 50 mM NaCl, the binding of the labelled nucleotide was about 2 fold increased by stimulation with saturating concentrations of neurotensin (EC50 value of 2.3±0.9 nM).
- The stimulation of [35S]-GTPγS binding by neurotensin was mimicked by the stable analogue of neurotensin, JMV-449 (EC50 value of 1.7±0.4 nM) and the neurotensin related peptide neuromedin N (EC50 value of 21±6 nM).
- The NT-induced [35S]-GTPγS binding was competitively inhibited by SR48692 (pA2 value of 9.55±0.28), a non-peptide neurotensin receptor antagonist. SR48692 alone had no effect on the specific binding of [35S]-GTPγS.
- The response to neurotensin was found to be inhibited by the aminosteroid U-73122, a putative inhibitor of phospholipase C-dependent processes, indicating that this drug may act at the G-protein level.
- Taken together, these results constitute the first characterization of the exchange of guanylyl nucleotides at the G-protein level that is induced by the neuropeptide neurotensin after binding to its receptor.
17.
Tiziano Croci Giulio Aureggi Luciano Manara Xavier Emonds-Alt Gerard Le Fur Jean-Pierre Maffrand Sylvain Mukenge Gianfranco Ferla 《British journal of pharmacology》1998,124(6):1321-1327
- Human in vitro preparations of transverse or distal colonic circular smooth muscle were potently and dose-dependently contracted by neurokinin A (EC50, 4.9 nM), the tachykinin NK2-receptor selective agonist [β-Ala8]neurokinin A (4–10) ([β-Ala8]NKA (4–10)) (EC50, 5.0 nM), neurokinin B (EC50, 5.3 nM) and substance P (EC50, 160 nM), but not by the tachykinin NK1-receptor selective agonist [Sar9Met(O2)11] substance P, or the NK3-receptor selective agonists, senktide and [MePhe7] neurokinin B. No regional differences between transverse and distal colon were observed in response to [β-Ala8]NKA (4–10).
- Atropine (1 μM) and tetrodotoxin (1 μM) did not significantly inhibit responses to [β-Ala8]NKA (4–10), neurokinin A, substance P or neurokinin B.
- The newly developed non-peptide antagonists for tachykinin NK2-receptors SR 48968, SR 144190 and its N-demethyl (SR 144743) and N,N-demethyl (SR 144782) metabolites, were used to challenge agonist responses, as appropriate. SR 144190 and the metabolites all potently and competitively antagonized the response to [β-Ala8]NKA (4–10), with similar potency (Schild plot pA2 values 9.4, 9.4 and 9.3, slope=1). SR 48968 antagonism was not competitive: the Schild plot slope was biphasic with a high (X intercept∼9.3) and a low (X intercept 8.4, slope 1.6) affinity site. Co-incubation of SR 48968 (10, 100 nM) and SR 144782 (10 nM) produced additive effects; in this experimental condition, SR 48968 apparent affinity (pKB) was 8.2. In addition, SR 144782 (0.1 μM) antagonized responses to neurokinin A, substance P and neurokinin B, with pKB consistent with its affinity for tachykinin NK2-receptors. The potent and selective NK1 and NK3-receptor antagonists, SR 140333 and SR 142801 (both 0.1 μM), failed to inhibit contractions induced by SP or NKB.
- In conclusion, the in vitro mechanical responses of circular smooth muscle preparations from human colon are strongly consistent with the presence of non-neuronal tachykinin NK2-receptors, but not tachykinin NK1- or NK3-receptors. Our findings with SR 48968 suggest the existence of two tachykinin NK2-receptor subtypes, that it seems to distinguish, unlike SR 144190 and its metabolites. However, the precise nature of SR 48968 allotopic antagonism remains to be elucidated, since allosteric effects at the tachykinin NK2-receptor might well account for the complexity of the observed interaction.
18.
M W Beukers C H W Klaassen W J De Grip D Verzijl H Timmerman R Leurs 《British journal of pharmacology》1997,122(5):867-874
- Rat histamine H2 receptors were epitope-tagged with six histidine residues at the C-terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope-tagged H2 receptor were prepared and were used to infect insect Sf9 cells.
- The His-tagged H2 receptors expressed in insect Sf9 cells showed typical H2 receptor characteristics as determined with [125I]-aminopotentidine (APT) binding studies.
- In Sf9 cells expressing the His-tagged H2 receptor histamine was able to stimulate cyclic AMP production 9 fold (EC50=2.1±0.1 μM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with Ki values of 0.60±0.43 μM, 0.25±0.15 μM and 28±7 nM, respectively (mean±s.e.mean, n=3).
- The expression of the His-tagged H2 receptors in infected Sf9 cells reached functional levels of 6.6±0.6 pmol mg−1 protein (mean±s.e.mean, n=3) after 3 days of infection. This represents about 2×106 copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol-β-cyclodextrin complex resulted in an increase of [125I]-APT binding up to 169±5% (mean±s.e.mean, n=3).
- The addition of 0.03 mM cholesterol-β-cyclodextrin complex did not affect histamine-induced cyclic AMP production. The EC50 value of histamine was 3.1±1.7 μM in the absence of cholesterol-β-cyclodextrin complex and 11.1±5.5 μM in the presence of cholesterol-β-cyclodextrin complex (mean±s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 μM histamine was identical, 85±18 pmol/106 cells in the absence and 81±11 pmol/106 cells in the presence of 0.03 mM cholesterol-β-cyclodextrin complex (mean±s.e.mean, n=3).
- Immunofluorescence studies with an antibody against the His-tag revealed that the majority of the His-tagged H2 receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well.
- These experiments demonstrate the successful expression of His-tagged histamine H2 receptors in insect Sf9 cells. The H2 receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His-tag reveal that only a limited amount of H2 receptor protein is functional. These functional receptors are targeted to the plasma membrane.
19.
Doreen Sarsero Peter Molenaar Alberto J Kaumann 《British journal of pharmacology》1998,123(3):371-380
- We have recently suggested the existence in the heart of a ‘putative β4-adrenoceptor'' based on the cardiostimulant effects of non-conventional partial agonists, compounds that cause cardiostimulant effects at greater concentrations than those required to block β1- and β2-adrenoceptors. We sought to obtain further evidence by establishing and validating a radioligand binding assay for this receptor with (−)-[3H]-CGP 12177A ((−)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one) in rat atrium. We investigated (−)-[3H]-CGP 12177A for this purpose for two reasons, because it is a non-conventional partial agonist and also because it is a hydrophilic radioligand.
- Increasing concentrations of (−)-[3H]-CGP 12177A, in the absence or presence of 20 μM (−)-CGP 12177A to define non-specific binding, resulted in a biphasic saturation isotherm. Low concentrations bound to β1- and β2-adrenoceptors (pKD 9.4±0.1, Bmax 26.9±3.1 fmol mg-1 protein) and higher concentrations bound to the ‘putative β4-adrenoceptor'' (pKD 7.5±0.1, Bmax 47.7±4.9 fmol mg−1 protein). In other experiments designed to exclude β1- and β2-adrenoceptors, (−)-[3H]-CGP 12177A (1–200 nM) binding in the presence of 500 nM (−)-propranolol was also saturable (pKD 7.6±0.1, Bmax 50.8±7.4 fmol mg−1 protein).
- The non-conventional partial agonists (−)-CGP 12177A (pKi 7.3±0.2), (±)-cyanopindolol (pKi 7.6±0.2), (−)-pindolol (pKi 6.6±0.1) and (±)-carazolol (pKi 7.2±0.2) and the antagonist (−)-bupranolol (pKi 6.6±0.2), all competed for (−)-[3H]-CGP 12177A binding in the presence of 500 nM (−)-propranolol at the ‘putative β4-adrenoceptor'', with affinities closely similar to potencies and affinities determined in organ bath studies.
- The catecholamines competed with (−)-[3H]-CGP 12177A at the ‘putative β4-adrenoceptor'' in a stereoselective manner, (−)-noradrenaline (pKiH 6.3±0.3, pKiL 3.5±0.1), (−)-adrenaline (pKiH 6.5±0.2, pKiL 2.9±0.1), (−)-isoprenaline (pKiH 6.2±0.5, pKiL 3.4±0.1), (+)-isoprenaline (pKi<1.7), (−)-RO363 ((−)-(1-(3,4-dimethoxyphenethylamino)-3-(3,4-dihydroxyphenoxy)-2-propranol)oxalate, pKi 5.5±0.1).
- The inclusion of guanosine 5-triphosphate (GTP 0.1 mM) had no effect on binding of (−)-CGP 12177A or (−)-isoprenaline to the ‘putative β4-adrenoceptor''. In competition binding studies, (−)-CGP 12177A competed with (−)-[3H]-CGP 12177A for one receptor state in the absence (pKi 7.3±0.2) or presence of GTP (pKi 7.3±0.2). (−)-Isoprenaline competed with (−)-[3H]-CGP 12177A for two states in the absence (pKiH 6.6±0.3, pKiL 3.5±0.1; % H 25±7) or presence of GTP (pKiH 6.2±0.5, pKiL 3.4±0.1; % H 37±6). In contrast, at β1-adrenoceptors, GTP stabilized the low affinity state of the receptor for (−)-isoprenaline.
- The specificity of binding to the ‘putative β4-adrenoceptor'' was tested with compounds active at other receptors. High concentrations of the β3-adrenoceptor agonists, BRL 37344 ((RR+SS)[4-[2-[[2-(3-chlorophenyl)-2-hydroxy - ethyl]amino]propyl]phenoxy]acetic acid, 6 μM), SR 58611A (ethyl{(7S)-7-[(2R)-2 - (3 - chlorophenyl) - 2 - hydroxyethylamino] - 5,6,7,8 - tetrahydronaphtyl2 - yloxy} acetate hydrochloride, 6 μM), ZD 2079 ((±)-1-phenyl-2-(2-4-carboxymethylphenoxy)-ethylamino)-ethan-1-ol, 60 μM), CL 316243 (disodium (R,R)-5-[2-[2-(3-chlorophenyl)-2-hydroxyethyl-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate, 60 μM) and antagonist SR 59230A (3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-2S-2-propanol oxalate, 6 μM) caused less than 22% inhibition of (−)-[3H]-CGP 12177A binding in the presence of 500 nM (−)-propranolol. Histamine (1 mM), atropine (1 μM), phentolamine (10 μM), 5-HT (100 μM) and the 5-HT4 receptor antagonist SB 207710 ((1-butyl-4-piperidinyl)-methyl 8-amino-7-iodo-1,4-benzodioxan-5-carboxylate, 10 nM) caused less than 26% inhibition of binding.
- Non-conventional partial agonists, the antagonist (−)-bupranolol and catecholamines all competed for (−)-[3H]-CGP 12177A binding in the absence of (−)-propranolol at β1-adrenoceptors, with affinities (pKi) ranging from 1.6–3.6 log orders greater than at the ‘putative β4-adrenoceptor''.
- We have established and validated a radioligand binding assay in rat atrium for the ‘putative β4-adrenoceptor'' which is distinct from β1-, β2- and β3-adrenoceptors. The stereoselective interaction with the catecholamines provides further support for the classification of the receptor as ‘putative β4-adrenoceptor''.
20.
Katia Varani Stefania Gessi Alessandro Dalpiaz Ennio Ongini Pier Andrea Borea 《British journal of pharmacology》1997,122(2):386-392
- The present study describes for the first time the characterization of the adenosine A2A receptor in human lymphocyte membranes with the new potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4 triazolo [1,5-c] pyrimidine, ([3H]-SCH 58261). In addition, both receptor affinity and potency of reference adenosine receptor agonists and antagonists were determined in binding and adenylyl cyclase studies.
- Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 0.85 nM and 35 fmol mg−1 protein, respectively. A series of adenosine receptor ligands were found to compete for the binding of 0.8 nM [3H]-SCH 58261 to human lymphocyte membranes with a rank order of potency consistent with that typically found for interactions with the A2A-adenosine receptor. In the adenylyl cyclase assay the same compounds exhibited a rank order of potency similar to that observed in binding experiments.
- Thermodynamic data indicate that [3H]-SCH 58261 binding to human lymphocytes is entropy and enthalpy-driven, a finding in agreement with the thermodynamic behaviour of antagonists for rat striatal A2A-adenosine receptors.
- It is concluded that in human lymphocyte membranes [3H]-SCH 58261 directly labels binding sites showing the characteristic properties of the adenosine A2A-receptor. The presence of A2A-receptors in peripheral tissue such as human lymphocytes strongly suggests an important role for adenosine in modulating immune and inflammatory responses.