Guinea Pig Brain Membranes Research Articles

Characterization of the δ-opioid receptor found in SH-SY5Y neuroblastoma cells

The δ-opioid receptor found in SH-SY5Y cells was characterized in terms of binding profile and ability to mediate the inhibition of forskolin-stimulated cAMP accumulation. Both DPDPE ([ d-Pen 2, d-Pen 5]enkephalin) and deltorphin II, compounds reported to be selective for the δ 1- and δ 2-opioid receptor respectively, were potent agonists in these cells. Binding studies indicated that naltrindole benzofuran (NTB) had significantly higher affinity than 7-benzylidenenaltrexone (BNTX); however, both compounds have high affinity for the δ-opioid receptor found in SH-SY5Y cells. Naltrindole benzofuran was found to be a potent antagonist, with an IC 50 of less than 1 nM, while 7-benzylidene naltrexone was found to be a relatively weak antagonist, requiring greater than 100 nM to inhibit 50% of agonist activity. Binding to intact SH-SY5Y cells was compared to binding to cell membranes and guinea-pig brain membranes. In each case, binding affinities were very similar. These studies suggest that the receptor found in SH-SY5Y cells could probably be classified as a δ 2-opioid receptor. However, the very similar binding characteristics of SH-SY5Y cells and guinea-pig brain membranes call into question the ability to label δ 1-opioid receptors. © Elsevier Science B.V. All rights reserved.

The Pharmacology of the Novel and Selective Sigma Ligand, PD 144418

The pharmacology of PD 144418 (1-propyl-5-(3-p-tolyl-isoxazol-5-yl)-1,2,3,6-tetrahydropyridine) was characterized using neurochemical, biochemical and behavioral techniques. For sigma (σ1 and σ2, respectively) sites, PD 144418 affinities were determined using whole guinea pig brain membranes with [3H](+)-pentazocine and neuroblastoma × glioma cell membranes using [3H]1,3-di-o-tolylguanidine (DTG) in the presence of 200 nM (+)-pentazocine. PD 144418 exhibited an affinity for σ1 of 0.08 nM (Ki) versus a Ki of 1377 nM for σ2 site. Additional receptor binding studies indicated that PD 144418 lacked affinity for dopaminergic, adrenergic, muscarinic and a variety of other receptors. In vitro studies indicated that PD 144418 reversed the N-methyl-d-aspartate (NMDA)-induced increase in cyclic GMP (cGMP) in rat cerebellar slices without affecting the basal levels, suggesting that σ1 sites may be important in the regulation of glutamine-induced actions. PD 144418 potentiated the decrease in 5-hydroxytryptophan caused by haloperidol in the mesolimbic region, but by itself had no effect in 5-hydroxytrypamine (5-HT) and dopamine (DA) synthesis. Behaviorally, similar to other σ ligands, PD 144418 antagonized mescaline-induced scratching at doses that did not alter spontaneous motor activity. This action is suggestive of potential antipsychotic property. It exhibited no anxiolytic and antidepressant properties in the models used. These results show that PD 144418 is a very selective σ1 agent, devoid of any significant affinity for other receptors and that σ1 site may modulate actions in the CNS. © 1997 Elsevier Science Ltd. All rights reserved.

Effects of Some 7-Arylidene and 7-Heteroarylidene Morphinan-6-ones on the Antinociceptive Activity of [d-Pen2, d-Pen5];enkephalin and [d-Ala2, Glu4]deltorphin II and on Multiple Opioid Receptors

Bhargava, H. N., G.-M. Zhao, J.-T. Bian, Y. Nan, S. P. Upadhyaya, X. Wei, W. J. Dunn, III and L. Bauer. Effects of some 7-arylidene and 7-heteroarylidene morphinan-6-ones on the antinociceptive activity of [d-Pen2, d-Pen5] enkephalin and [d-Ala2, Glu4]deltorphin II and on multiple opioid receptors. peptides 18(5) 695–701, 1997.—The in vivo and functional effects of several 7-arylidene and 7-heteroarylidene morphinan-6-ones were determined at the μ-, δ-, and κ-opioid receptors using the guinea pig brain membranes, guinea pig ileum (GPI), and mouse vas deferens (MVD). In vivo effects included the antagonism by these compounds given subcutaneously on the antinociceptive actions of intracerebroventricularly injected [d-Pen2, d-Pen5]enkephalin (DPDPE) and [d-Ala2, Glu4]deltorphin II (deltorphin II), the highly selective putative δ1- and δ2- opioid receptor agonists. Finally, the partition coefficients of these compounds were estimated (CLOGP) and determined experimentally at pH 7.4 in the 1-octanol/water system. Compared with E-7-benzylidenenaltrexone (BNTX), most compounds except for E-7-(4-chlorobenzylidene)naltrexone, were more potent at δ-opioid receptors than at the μ-opioid receptor, whereas, in comparison to the κ-opioid receptor, the activities of the E-7-arylidene or E-7-heteroarylidene naltrexone derivatives at the δ-receptor were in the following order, where the 7-substituents were: 4-fluorobenzylidene- > benzylidene > 3-pyridylmethylene- > 4-pyridylmethylene- > 1-methyl-2-imidazolylmethylene > 4-chlorobenzylidene. In the MVD preparation, the potencies at the δ-opioid receptor, in comparison to BNTX, were in the following order, where the 7-substituents were: benzylidene = 1-methyl-2-imidazolylmethylene- > 4-fluorobenzylidene- = 3-pyridylmethylene- = 4-pyridylmethylene-. All compounds antagonized δ1 and δ2-opioid receptor agonist-induced analgesia. The antagonist potencies at the δ1-opioid receptor were in the following order, where the 7-substituents were: benzylidene- > 4-chlorobenzylidene- > 4-fluorobenzylidene- > 3-pyridylmethylene- > 1-methyl-2-imidazolylmethylene- ≈ 4-pyridylmethylene-, whereas at the δ2-opioid receptor, the order was benzylidene- > 4-chlorobenzylidene- > 4-fluorobenzylidene- > 3-pyridylmethylene- > 1-methyl-2-imidazolylmethylene- > 4-pyridylmethylene. In general, all compounds exhibited greater potency at the δ2- than δ1-opioid receptor. The computed partition coefficients were, as expected, greater than the apparent log P values, which were determined experimentally. Generally, the lipophilicity values in decreasing order were: 4-chlorobenzylidene- > 4-fluorobenzylidene- > benzylidene > 3-pyridylmethylene- = 4-pyridylmethylene- > 1-methyl-2-imidazolylmethylene-. In general, the benzylidene and 4-pyridylmethylene derivatives, which have medium lipophilicities, were equally effective at the δ1- and δ2- receptors; the 3-pyridylmethylene and 1-methyl-2-imidazolylmethylene derivatives had lower lipophilicities and were more selective for the δ2- than δ1- receptor; the 4-chlorobenzylidene and 4-fluorobenzylidene derivatives were more lipophilic and had intermediate activity. The plot of pED50 values for the in vivo tests for the δ1- and δ2- receptors showed that the two receptors are not independent with respect to this series of compounds.

Comparison of binding parameters of σ1 and σ2 binding sites in rat and guinea pig brain membranes: novel subtype-selective trishomocubanes

Comparisons of binding parameters of [ 3H](+)-pentazocine and [ 3H]1,3-di- o-tolylguanidine (DTG) at σ binding sites in guinea pig and rat brain membranes demonstrated that [ 3H](+)-pentazocine binds to a single high-affinity site, whereas [ 3H]DTG binds to two high-affinity sites in both species. The K d values of the radioligands were similar in both types of membranes. However, the density of σ 1 sites in guinea pig was significantly higher than that of rat. Novel trishomocubanes were tested for their affinities at σ 1 and σ 2 binding sites in guinea pig brain membranes using [ 3H](+)-pentazocine and [ 3H]DTG as the radioligands. N-(4-Phenylbutyl)-3-hydroxy-4-azahexacyclo[5.4.1.0 2,6.0 3,10.0 5,9.0 8,11]dodecane (ANSTO-14) showed the highest affinity for the σ 1 site ( K i = 9.4 nM) and 19-fold σ 1 σ 2 selectivity, as a result of increasing the alkyl chain between the cubane moiety and the aromatic ring. N-(3′-Fluorophenyl)methyl-3-hydroxy-4-azahexacyclo[5.4.1.0 2,6.0 3,10.0 5,9.0 8,11]dodecane (ANSTO-19), displayed the highest affinity for σ 2 sites ( K i = 19.6 nM) and 8-fold σ 2 σ 1 selectivity due to a fluoro substitution in the meta position of the aromatic ring. These represent structurally novel lead compounds, especially for the development of selective σ 2 receptor ligands.

Synthesis, in vitro validation and in vivo pharmacokinetics of [ 125I]N-[2-(4-Iodophenyl)Ethyl]- N-Methyl-2-(1-Piperidinyl) ethylamine: A high-affinity ligand for imaging sigma receptor positive tumors

N-[2-(4-iodophenyl)ethyl]- N-methyl-2-(1-piperidinyl)ethylamine, IPEMP, and the corresponding bromo derivative, BrPEMP, have been synthesized and characterized. Both BrPEMP and IPEMP were evaluated for sigma-1 and sigma-2 subtype receptor affinities and found to possess very high affinities for both receptor subtypes. The precursor for radioiodination n-tributylstannylphenylethylpiperidinylethylamine was prepared from its bromo derivative by palladium-catalyzed stannylation reaction. Radioiodinated 4-[ 125I]PEMP was readily prepared in high yields and high specific activity by oxidative iododestannylation reaction using chloramine-T as oxidizing agent. Sites labeled by 4-[ 125I]PEMP in guinea pig brain membranes showed high affinity for BD1008, haloperidol, and (+)-pentazocine (Ki = 5.06 ± 0.40, 32.6 ± 2.75, and 48.1 ± 8.60 nM, respectively), which is consistent with sigma receptor pharmacology. Competition binding studies of 4-[ 125I]PEMP in melanoma (A375) and MCF-7 breast cancer cells showed a high affinity, dose-dependent inhibition of binding with known sigma ligand N-[2-(3,4-dichlorophenyl)ethyl]- N-methyl-2-(1-pyrrolidinyl) ethylamine, BD1008 (Ki = 5, 11 nM, respectively), supporting the labeling of sigma sites in these cells. Haloperidol, however showed a weaker (Ki = 100–200 nM) affinity for the sites labeled by 4-[ 125I]PEMP in these cells. Biodistribution studies of 4-[ 125I]PEMP in rats showed a fast clearance of this radiopharmaceutical from blood, liver, lung, and other organs. A co-injection of 4-IPEMP with 4-[ 125I]PEMP resulted in 37%, 69%, and 35% decrease in activity in liver, kidney, and brain (organs possessing sigma receptors), respectively at 1-h postinjection. These results suggest that 4-[ 125I]PEMP is a promising radiopharmaceutical for pursuing further studies in animal models with tumors.

Binding properties of SA4503, a novel and selective σ1 receptor agonist

The binding profiles of SA4503 (1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride), a novel σ receptor ligand, to σ 1 and σ 2 receptor subtypes in guinea pig and rat brain membranes were evaluated. SA4503 showed a high affinity for the σ 1 receptor subtype labeled by (+)-[ 3H]pentazocine (IC 50 = 17.4 ± 1.9 nM), while it had about 100-fold less affinity for the σ 2 receptor subtype labeled by [ 3H]1,3-di(2-tolyl)guanidine ([ 3H]DTG) in the presence of 200 nM (+)-pentazocine. SA4503 showed little affinity for 36 other receptors, ion channels and second messenger systems. The inhibition curves of SA4503 for (+)-[ 3H]pentazocine binding were shifted to the right in the presence of guanosine 5′- o-(3-thiotriphosphate) (GTPγS), as similar to those of (+)-3-(3-hydroxyphenyl)- N-(1-propyl)piperidine ((+)-3-PPP) and (+)-pentazocine, σ 1 receptor agonists. SA4503 significantly increased the K D value, but did not affect the B max value for specific (+)-[ 3H]pentazocine binding. These results indicated that SA4503 is a potent and selective agonist for the σ 1 receptor subtype in the brain. In addition, SA4503 inhibited specific (+)-[ 3H]pentazocine binding in a competitive manner.

P-10-22 - CCK-4-induced calcium mobilization in T cells is enhanced in panic disorder

This chapter describes the radioligand binding assay methods used to identify and characterize the interaction of agents with the cholecystokinin-A (CCK-A) and CCK-B/gastrin receptor subtypes. The chapter also discusses the biochemical and pharmacological properties of nonpeptide CCK-A and CCK-B/gastrin antagonists, and their utility as radioligands. Rat pancreas and guinea pig cerebral cortex are frequently used as sources for CCK-A and CCK-B receptors, respectively. In a study described in the chapter, CCK-B/gastrin receptor antagonism was examined in vivo using the in situ perfused stomach preparation. The results of the study suggested that, as proposed for the cholinergic receptor or other receptor systems, the CCK-A receptor may have either two classes of binding sites or two conformational states that have different affinities for agonists but the same affinities for antagonists. The CCK receptor agonists, including CCK-8, CCK-8 desulfate, gastrin, and CCK-4, are also effective in inhibiting specific [3H]L-365,260 binding to guinea pig brain membranes. Removal of MgCl2 or addition of the guanyl nucleotide GppNHP results in a selective reduction of [125I]CCK-8 binding and [3H]L-365,260 binding differently.

Cholecystokinin and gastrin are not equally sensitive to GTPγS at CCK B receptors: importance of the sulphated tyrosine

We have shown that gastrin and cholecystokinin octapeptide (CCK-8) are differently coupled to G protein (GTP-binding protein) through type B cholecystokinin receptors in guinea-pig brain membranes and Jurkat cells. Indeed, the gastrin-13 binding affinity is strongly reduced by stable guanyl nucleotides, whereas CCK-8 binding is only slightly affected. In order to determine the structural requirements regulating such coupling, we have synthesized several gastrin and cholecystokinin fragments (sulphated or unsulphated) elongated at the N-terminus of the common C-terminal tetrapeptide. We investigated their interaction with CCK B receptors in guinea pig brain membranes and Jurkat cells and their involvement in the G protein coupling. Their apparent binding affinities to CCK B receptors were measured by inhibition of [ 125I]Bolton Hunter-CCK-8 (3-[ 125I]iodo-4-hydroxyphenyl)propionyl-CCK-8) binding in the presence or absence of GTP γS (guanosine 5′- O-(3-thio)triphosphate) or aluminium tetrafluoride (AlF 4 −). Activation of the G proteins by GTP γS or AlF 4 − led to a decrease in binding affinity for the gastrin related peptides, the common CCK-gastrin C-terminal forms, the cholecystokinin hexapeptide and the unsulphated cholecystokinin heptapeptide. Sulphated CCK-7, CCK-8, and cionin apparent binding affinities were not affected. These finding indicated that the sulphated tyrosine in position 7 in CCK (as counted from the C-terminus), provides the cholecystokinin selectivity for the CCK B receptor compared to gastrin. The results are discussed with the aim to better clarify the physiological relevance of gastrin and cholecystokinin toward CCK B receptors and their related intracellular events.

Suppression of eosinophil function by RP 73401, a potent and selective inhibitor of cyclic AMP-specific phosphodiesterase: comparison with rolipram.

1. We have investigated the inhibitory potency of RP 73401, a novel, highly selective and potent inhibitor of cyclic AMP-specific phosphodiesterase (PDE IV), against partially-purified PDE isoenzymes from smooth muscle and the particulate PDE IV from guinea-pig eosinophils. The inhibitory effects of RP 73401 on the generation of superoxide (.O2-), major basic protein (MBP) and eosinophil cationic protein (ECP) from guinea-pig eosinophils have also been studied. 2. RP 73401 potently inhibited partially-purified cyclic AMP-specific phosphodiesterase (PDE IV) from pig aortic smooth muscle (IC50 = 1.2 nM); it was similarly potent against the particulate PDE IV from guinea-pig peritoneal eosinophils (IC50 = 0.7 nM). It displayed at least a 19000 fold selectivity for PDE IV compared to its potencies against other PDE isoenzymes. Rolipram was approximately 2600 fold less potent than RP 73401 against pig aortic smooth muscle PDE IV (IC50 = 3162 nM) and about 250 times less potent against eosinophil PDE IV (IC50 = 186 nM). 3. Solubilization of the eosinophil particulate PDE IV increased the potency of rolipram 10 fold but did not markedly affect the potency of RP 73401. A similar (10 fold) increase in the PDE IV inhibitory potency of rolipram, but not RP 73401, was observed when eosinophil membranes were exposed to vanadate/glutathione complex (V/GSH). 4. Reverse transcription polymerase chain reaction (RT-PCR), using primer pairs designed against specific sequences in four distinct rat PDE IV subtype cDNA clones (PDE IVA-D), showed only mRNA for PDE IVD in guinea-pig eosinophils. PDE IVD was also the predominant subtype expressed in pig aortic smooth muscle cells. 5. RP 73401 (Kiapp = 0.4 nM) was 4 fold more potent than (+/-)-rolipram (Kiapp = 1.7 nM) in displacing[3H]-(+/-)-rolipram from guinea-pig brain membranes.6. In intact eosinophils, RP 73401 potentiated isoprenaline-induced cyclic AMP accumulation(EC50 = 79 nM). RP 73401 also inhibited leukotriene B4-induced generation of *02- (IC50 = 25 nM), and the release of major basic protein (ICo = 115 nM) and eosinophil cationic protein (IC50 = 7 nM). Rolipram was 3-14 times less potent than RP 73401.7. Thus RP 73401 is a very potent and selective PDE IV inhibitor which suppresses eosinophil function suggesting that it may be a useful agent for the treatment of inflammatory diseases such as asthma. The greatly different inhibitory potencies of rolipram against PDE IV from smooth muscle and eosinophils(in contrast to the invariable effects of RP 73401) are unlikely to be attributable to diverse PDE IV subtypes but suggest distinct interactions of the two inhibitors with the enzyme.

Characteristics of binding of [3H]NE-100, a novel sigma-receptor ligand, to guinea-pig brain membranes

We examined the characteristics of binding of radiolabeled N,N-dipropyl-2-[4-methoxy-3-(2-phenyl-ethoxy)phenyl]- ethylamine monohydrochloride ([3H] NE-100), a highly potent and selective sigma-receptor ligand, to guinea-pig brain membranes. [3H]NE-100 showed saturable and reversible binding to sigma binding sites. A dissociation constant (Kd) and maximal numbers of binding sites (Bmax) obtained from Scatchard plot analysis were 1.2 +/- 0.1 nM and 1049.3 +/- 115.1 fmol/mg protein (n = 3), respectively. NE-100 was the most potent inhibitor of [3H]NE-100 binding among several structurally dissimilar sigma-receptor ligands, including haloperidol and (+)-pentazocine. (+)-Benzomorphanes had more than a 10-fold potent inhibitory activity over (-)-benzomorphanes, with regard to [3H]NE-100 binding. The binding of [3H]NE-100 was not influenced by histaminergic, dopaminergic, adrenergic, serotonergic cholinergic or glutaminergic agents at 10(-7) M. GTP-gamma-S and phenytoin also did not affect the binding of [3H]NE-100. A higher [3H]NE-100 binding was observed in the cerebellum and medulla oblongata. Except for the nuclear fraction, the highest level of [3H]NE-100 binding to subcellular fractions was observed in microsomal fractions. These results suggest that NE-100 selectively binds, with a high affinity, to sigma-1 binding sites in guinea-pig brain membranes, as an "antagonist".

Opioid peptide receptor studies. 3. Interaction of opioid peptides and other drugs with four subtypes of the kappa 2 receptor in guinea pig brain.

Using guinea pig, rat, and human brain membranes depleted of mu and delta receptors by pretreatment with the site-directed acylating agents BIT (mu selective) and FIT (delta selective), previous studies from our laboratory resolved two subtypes of the kappa 2 binding site, termed kappa 2a and kappa 2b. In more recent studies, we used 6 beta-[125Iodo]-3,14-dihydroxy-17-cyclopropylmethyl-4,5 alpha-epoxymorphinan ([125I]IOXY) to characterize multiple kappa 2 binding sites in rat brain. The results indicated that [125I]IOXY, like [3H]bremazocine, selectively labels kappa 2 binding sites in rat brain membranes pretreated with BIT and FIT. In the rat brain, using 100 nM [D-Ala2-MePhe4,Gly-ol5]enkephalin to block [125I]IOXY binding to the kappa 2b site, we resolved two subtypes of the kappa 2a binding site. In the present study we examined the binding of [125I]IOXY to the kappa 2 receptors of guinea pig brain. As observed in rat brain, [125I]IOXY, under appropriate assay conditions, selectively labels kappa 2 binding sites. Quantitative binding studies readily demonstrated the presence of kappa 2a and kappa 2b binding sites. The kappa 2a binding sites were selectively assayed using 5 microM [Leu5]enkephalin to block [125I]IOXY binding to the kappa 2b sites, and kappa 2b sites were selectively assayed using 5 microM (-)-(1S,2S)-U50,488 to block [125I]IOXY binding to the kappa 2a sites. Under these conditions, two subtypes of the kappa 2a site were resolved with high (kappa 2a-1) and low (kappa 2a-2) affinity for nor-BNI (Ki values = 0.88 and 476 nM) and CI977 (Ki values = 17.5 and 95,098 nM). Similarly, two subtypes of the kappa 2b site were observed with high (kappa 2b-1) and low (kappa 2b-2) affinity for [D-Ala2-MePhe4,Gly-ol5]enkephalin (DAMGO) (Ki values = 97 and 12,321 nM) and alpha-neoendorphin (Ki values = 33 and 5308 nM). Two-site models were also resolved in the presence of 100 microM 5'-guanylyimidodiphosphate (GppNHp). We carried out detailed ligand selectivity analysis of the multiple kappa 2 binding sites. Most test agents were either nonselective or selective for the kappa 2a-1 site. Nalbuphine was moderately selective for the kappa 2a-2 site. Similarly, although most test agents were either nonselective or selective for the kappa 2b-1 site, butorphanol, and the delta antagonists naltrindole, naltriben, and 7-benzylidene-7-dehydronaltrexone were moderately selective for the kappa 2b-2 site.(ABSTRACT TRUNCATED AT 400 WORDS)

Isothiocyanate-substituted kappa-selective opioid receptor ligands derived from N-methyl-N-[(1S)-1-phenyl-2-(1-pyrrolidinyl)ethyl] phenylacetamide.

The synthesis of isothiocyanate-substituted kappa-selective opioid ligands derived from N-methyl-N-[(1S)-1-phenyl-2-(1-pyrrolidinyl)ethyl]ethyl]phenylacetami de (8) and their effects in radioligand displacement assays are reported. Ligands 3-5 with the S-absolute configuration were prepared with the isothiocyanate functionality at the 2-, 3-, and 4-positions in the phenylacetamide aromatic ring. The 2-isothiocyanato-4,5-dichlorophenylacetamide 6 was prepared to evaluate the effect of 4,5-dichloro substitution in the same aromatic ring as the 2-isothiocyanate function. N-Methyl-N-[(1S)-1-(4-isothiocyanatophenyl)-2-(1-pyrrolidinyl) ethyl]-3,4-dichlorophenylacetamide (7), with the 4-isothiocyanate function in the 1-phenyl ring, was prepared for comparison with the other compounds in the series. Of the prepared ligands, 7 and 8 (IC50s congruent to 1.4-1.8 nM) were approximately equal in affinity with 2 (ICI-199,441), followed by 3 and 6. All of these compounds were more kappa-selective than 2, as well. The binding characteristics of 8 show that the previously reported 4,5-dichloro substitution is not required for high affinity and kappa-selectivity. All of the synthesized isothiocyanate-substituted ligands irreversibly inhibited radioligand binding to guinea pig brain membrane preparations, including compound 2 (ICI-199,441) which had no isothiocyanate functionality.

Pharmacological properties of R-84760, a novel kappa-opioid receptor agonist.

The pharmacological properties of a structurally novel kappa-opioid receptor agonist, (3R)-3-(1-pyrrolidinylmethyl)-4-[(1S)-5,6-dichloro-1-indancarbo nyl]- tetrahydro-1,4-thiazine hydrochloride (R-84760), were examined. In an opioid receptor binding assay with a guinea pig brain membrane fraction, R-84760 showed a 5 times higher affinity for the kappa-opioid receptor than CI-977, the most potent reference kappa-opioid receptor agonist, and selectivity of R-84760 for the kappa-opioid receptor was higher than that of U-69593, a highly selective kappa-opioid receptor agonist. R-84760 was functionally 2.5 times more potent than CI-977 as a kappa-opioid receptor agonist in rabbit vas deferens. Subcutaneously administered R-84760 had an antinociceptive effect in the phenylquinone writhing test in mice and its potency was 5-846 times higher than those of CI-977, U-50488, morphine, pentazocine and butorphanol. On oral administration, R-84760 was 20-2077 times more potent than the same reference drugs. The antinociceptive effect of R-84760 was not antagonized by naloxone in a dose at which naloxone antagonized the effect of morphine, but on the other hand nor-binaltorphimine antagonized the effect of R-84760 at a dose which did not affect the effect of morphine. R-84760 and the reference kappa-opioid receptor agonists produced sedative and diuretic effects in mice with the same order of potency as for the antinociceptive effect. Tolerance to the antinociceptive effect of R-84760 barely developed, and naloxone-induced jumping tests for morphine-like physical dependence of R-84760 gave negative results.

Gastrin 13 and the C-terminal octapeptide of cholecystokinin are differently coupled to G-proteins in guinea-pig brain membranes

In the course of our study concerning gastrin and cholecystokinin (CCK) receptors, we synthesized and characterized a labelled gastrin ligand, [ 125I]BH[Leu 15]gastrin-(5–17) (3-(3-[ 125I]iodo-4-hydroxyphenyl)propionyl[Leu 15]gastrin-(5–17)). On isolated canine fundic mucosal cells and human Jurkat lymphoblastic cell line, known to express CCK B/gastrin receptors, the binding experiments performed indicate that [ 125I]BH[Leu 15]gastrin-(5–17) provides a convenient biologically active ligand for cholecystokinin/gastrin receptor studies. We showed in this study that, on guinea-pig brain membranes known to possess CCK B and CCK A receptors, [ 125I]BH[Leu 15]gastrin-(5–17) binds to a single class of high-affinity binding sites in a saturable and specific manner. [ 125I]BH[Leu 15]gastrin-(5–17) interacts with guinea-pig brain membranes with a maximal binding capacity that is about three-fold lower than that of [ 125I]BHCCK 8 (CCK 8, the C-terminal octapeptide of cholecystokinin). The apparent affinities of CCK analogoues and selective CCK receptor antagonists L-365,260 and MK-329 for the sites labelled by both probes were in accordance with a CCK B-like profile. Association-dissociation kinetics of [ 125I]BH[Leu 15]gastrin-(5–17) and [ 125I]BHCCK 8 were performed and compared. They showed that [ 125I]BHCCK 8 equilibrated more slowly than [ 125I]BH[Leu 15]gastrin-(5–17). The effects of pH, monovalent and divalent cations on binding of both probes were investigated. The results obtained did not indicate strong differences between [ 125I]BH[Leu 15]gastrin-(5–17) and [ 125I]BHCCK 8 binding. Binding experiments in the presence of stable analogues of GTP showed a different behaviour between [ 125I]BH[Leu 15]gastrin-(5–17) and [ 125I]BHCCK 8. GTPγS strongly decreased [ 125I]BH[Leu 15]gastrin-(5–17) binding whereas it weakly affected [ 125I]BHCCK 8 binding. The 5′-adenylylimidodiphosphate was found to exert a similar effect than GTPγS on gastrin and CCK 8 binding. The results of binding studies carried out with [ 125I]BH[Leu 15]gastrin-(5–17) showed that gastrin binds specifically to guinea-pig brain membranes. Furthermore, the different effects of guanyl nucleotides on gastrin and CCK binding strongly suggested that gastrin and CCK trigger a differential G protein coupling through the same binding sites.

Receptor binding profiles of KB-5492, a novel anti-ulcer agent, at σ receptors in guinea-pig brain

We studied the receptor binding profile of 4-methoxyphenyl 4-(3,4,5-trimethoxybenzyl)-1-piperazine acetate monofumarate monohydrate (KB-5492), a novel anti-ulcer agent, for the σ receptor in guinea-pig brain membranes. KB-5492 selectively inhibited specific [ 3H]1,3-di(2-tolyl)guanidine (DTG) binding to the σ receptor ( IC 50 = 3.15 μ M ) with a pseudo-Hill coefficient of 0.3. Computer-assisted analysis revealed that KB-5492 bound to high- and low-affinity sites. Although KB-5492 had weak affinity for α 2- adrenoceptors at 10 μM, it was almost inactive at a concentration of 10 μM in 33 other binding assays for receptors, second messenger system and ion channels. σ Receptor ligands such as haloperidol, DTG, (+)-3-(3-hydroxyphenol)- N-(1-propyl)piperidine (3-PPP), rimcazole and (−)-3-PPP inhibited specific [ 3H]DTG binding and their IC 50 values were 0.003, 0.044, 0.33, 0.67 and 1.03 μM, respectively. On the other hand, various anti-ulcer agents such as cetraxate, cimetidine, omeprazole, sofalcone, sucralfate, teprenone and troxipide could hardly displace specific [ 3H]DTG binding at 100 μM. Scatchard-Rosenthal analysis indicated that [ 3H]DTG bound to a single site, and K D and B max values for [ 3H]DTG were 87 .3 nM and 679.3 fmol/mg protein, respectively. KB-5492 significantly decreased the B max value, but did not affect the K D value . In contrast, haloperidol and DTG significantly increased the K D values, but did not affect the B max values. These findings indicate that KB-5492 selectively bound to the [ 3H]DTG-labeled σ receptor and that other anti-ulcer agents had little affinity for the σ receptor. Furthermore, KB-5492 inhibited specific [ 3H]DTG binding in a non-competitive manner.

Non-peptide angiotensin receptor antagonists bind to tachykinin NK 3 receptors of rat and guinea pig brain

[ 3H]Senktide, a highly selective tachykinin NK 3 receptor agonist, was used to study tachykinin NK 3 receptors of rat and guinea pig brain. Guinea pig brain membranes had a K d of 3.9 ± 0.5 nM and a B max of 42 fmol/mg. Dose-displacement experiments with neurokinins and selective tachykinin receptor agonists revealed the following order of potency: [MePhe 7]neurokinin B > neurokinin B > substance P > neurokinin A. This order is typical for a tachykinin NK 3 receptor. To further characterize the specificity of this receptor, the effects of unrelated compounds such as: bradykinin, angiotensin II, bombesin and their structural analogs were also evaluated on the binding of [ 3H]senktide. Unexpectedly, the angiotensin AT 1 receptor antagonists DuP 753 (2- n-butyl-4-chloro-5-hydroxymethyl-1[2′-(1 H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole potassium salt), L-158,809 (5,7-dimethyl-2-ethyl-3-[2′-(1 H-tetrazol-5-yl) [1,1′-biphenyl-4-yl) methyl]-3 H-imidazo[4,5-β]pyridine H 2O) and EXP 3174 (2- n-butyl-4-chloro-1-[2′-(1 H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole-5-carboxylic acid), inhibited the binding of [ 3H]senktide to its receptor in the guinea pig brain membranes with IC 50 values of 18 μM, 25 μM and 50 μM, respectively. Similar effects were also observed with rat brain membranes. Angiotensin II, saralasin ([Sar 1,Val 5,Ala 8]angiotensin II, a peptide angiotensin AT 1 receptor antagonist) and PD 123,319 (1-[4-(dimethylamino)3-methylaphenyl]methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1 H imidazo[4,5- c]pyridine-6-carboxylic acid, a known non-peptide angiotensin AT 2 receptor antagonist) did not inhibit the binding of [ 3H]senktide to either type of membrane. Furthermore DuP 753, L-158,809 and EXP 3174 did not show any effect on the binding of the tachykinin NK 1 selective receptor agonist, [ 3H][Sar 9, Met (O) 2 11]SP, to guinea pig and rat brain membranes, thus eliminating a possible interaction of these angiotensin AT 1 receptor antagonists with the tachykinin NK 1 receptors. These studies constitute the first evidence of a non-selective interaction of DuP 753, L-158,809 and EXP 3174 with tachykinin NK 3 receptors.

Neurotensin receptor interaction with dopaminergic systems in the guinea-pig brain shown by neurotensin receptor antagonists

Neurotensin has been suggested to be involved in neurological and mental disorders associated with altered dopaminergic transmission. The lack of a potent neurotensin receptor antagonist had prevented us from studying the real physiological implication of this peptide in brain function. We thus recently developed such a non-peptide neurotensin receptor antagonist, SR 48692, (2-(1-(7-chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1 H-pyrazole-3-carbonyl)amino)-adamantane-2-carboxylic acid), which appeared to be potent in various central and peripheral preparations. In the present study, we tested the pharmacological properties of SR 48692 and of two optically synthetic analogs of this compound on neurotensin binding to both adult guinea-pig brain membrane homogenates and coronal brain sections, as well as on neurotensin stimulation of the K +-evoked release of [ 3H]dopamine in guinea-pig striatal slices. Our results demonstrated that (1) high-affinity neurotensin binding sites are present in the guinea-pig brain in regions rich in both dopamine cell bodies and terminals; (2) the binding of neurotensin is inhibited by SR 48692 and its related S(+) active analog, SR 48527, with IC 50 values in the nM range and (3) the non-peptide antagonist has no agonist effect but antagonizes neurotensin-induced [ 3H]dopamine release from guinea-pig striatal nerve terminals.

Gastrin13 binds to CCKB brain membrane receptors coupled to G protein in guinea pig brain membranes.

Gastrin13 binds to CCKB brain membrane receptors coupled to G protein in guinea pig brain membranes.

Low affinity of FMRFamide and four FaRPs (FMRFamide-related peptides), including the mammalian-derived FaRPs F-8-Famide (NPFF) and A-18-Famide, for opioid μ, δ, [formula omitted], [formula omitted], or [formula omitted] receptors

The binding affinities at opioid receptor subtypes in rat or guinea pig brain membranes were determined for the neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH 2), the two mammalian-derived FMRFamide-related peptides F-8-Famide (NPFF; Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH 2) and A-18-Famide (Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-NH 2), and the two other FMRFamide-related peptides Tyr-Phe-Met-Arg-Phe-NH 2 (Tyr-FMRFamide) and Pro-Gln-Arg-Phe-NH 2 (Pro-Gln-RFamide). The μ and δ sites were labeled in rat brain membranes using tritiated [ d-Ala 2, N- MePhe 4 ,Gly-ol 5]enkephalin ([ 3H]DAMGO) and [ d-Ala 2, d-Leu 5]enkephalin ([ 3H]DADLE), respectively. The κ sites were labeled in guinea pig brain using [ 3H]U-69,593 after treatment with BIT and FIT for κ 1 and [ 3H]bremazocine after pretreatment with BIT and FIT for κ 2 . The κ 2 a binding sites were assayed using [Leu 5]enkephalin to block κ 2 b sites and the κ 2 b sites were assayed using (−)-(1 S,2 S)-U50,488 to block κ 2 a sites. Neither FMRFamide nor any of the FMRFamide-related peptides (up to 61.0 μM) displayed significant affinity at any of the subtypes of opioid receptor. Hence, the known ability of FMRFamide and FaRPs to interact with the opioid system does not appear to be related to direct binding to these opioid receptors.

RTI-4793-14, a new ligand with high affinity and selectivity for the (+)-MK801-insensitive [3H]1-]1-(2-thienyl)cyclohexyl]piperidine binding site (PCP site 2) of guinea pig brain.

[3H]TCP, an analog of the dissociative anesthetic phencyclidine (PCP), binds with high affinity to two sites in guinea pig brain membranes, one that is MK-801 sensitive and one that is not. The MK-801-sensitive site (PCP site 1) is associated with NMDA receptors, whereas the MK-801-insensitive site (PCP site 2) may be associated with biogenic amine transporters (BAT). Although several "BAT ligands" are known that bind selectively to PCP site 2 and not to PCP site 1 (such as indatraline), these compounds have low affinity for site 2 (Ki values > 1 microM). Here we demonstrate that the novel pyrrole RTI-4793-14 is a selective, high affinity ligand for PCP site 2. We determined the IC50 values of RTI-4793-14 and several reference compounds [PCP, (+)-MK801 and indatraline] for PCP site 1 (assayed with [3H](+)-MK801), PCP site 2 (assayed with [3H]TCP in the presence of 500 nM (+)-MK801) and a variety of BAT-related measures ([3H]CFT binding to the DA transporter, [3H]nisoxetine binding to the norepinephrine transporter, [3H]dopamine uptake, [3H]serotonin uptake). In addition, we determined the ability of RTI-4793-14 to block NMDA responses in cultured hippocampal neurons under voltage clamp. (+)-MK801 had high affinity for PCP site 1 (4.6 nM) and potently inhibited NMDA-induced responses, but was much less potent in the BAT-related measures (IC50 s > 10 microM). PCP had high affinity at PCP site 1 (IC50 = 92 nM) and PCP site 2 (IC50 = 117 nM), and was moderately potent in all BAT-related measures except [3H]nisoxetine binding.(ABSTRACT TRUNCATED AT 250 WORDS)