Characterization of the binding of [3H]L-365,260: a new potent and selective brain cholecystokinin (CCK-B) and gastrin receptor antagonist radioligand

[3H]L-365,260, [(3R-(+)-2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4- benzodiazepin-3-yl)-N'-(3-methylphenyl)urea], a new potent and selective nonpeptide brain cholecystokinin (CCK-B) and gastrin receptor antagonist, bound saturably and reversibly to guinea pig brain membranes. Scatchard analysis indicated a single class of high affinity (Kd = 2.3 nM) binding sites. The binding of [3H]L-365,260 was stereospecific, because unlabeled L-365,260 (an R-enantiomer) was approximately 100 times more potent than its S-enantiomer in displacing binding. The relative potencies of various CCK/gastrin-related peptides and nonpeptide peripheral CCK-A antagonists in displacing [3H]L-365,260 brain binding correlated with their potencies in displacing the binding of 125I-CCK to brain receptors but not their potencies in displacing the peripherally selective CCK-A ligand [3H]L-364,718 from pancreatic receptors. The regional distribution of [3H]L-365,260 binding in various brain areas correlated with 125I-CCK binding. Specific [3H]L-365,260 binding to guinea pig brain membranes was reduced by omission of NaCl but was not affected by omission of MgCl2 or addition of guanosine 5'-(beta-gamma-imido)triphosphate or various pharmacological agents known to interact with other common peptide and nonpeptide receptor systems. [3H]L-365,260 also bound in a specific manner to guinea pig gastric glands but only negligibly to guinea pig or rat pancreas. The binding of [3H]L-365,260 to gastric glands was inhibited by CCK/gastrin antagonists with potencies similar to those for inhibition of 125I-gastrin binding in this tissue. Collectively, the data indicates that [3H]L-365,260 represents a new potent nonpeptide antagonist radioligand suitable for the study of brain CCK-B and gastrin receptors.     

Characterization of [3H](+/-)L-364,718 binding to solubilized cholecystokinin (CCK) receptors of rat pancreas

The binding of [3H](+/-)L-364,718 (3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl )-1H-indole-2-carboxamide), an extremely potent nonpeptide cholecystokinin (CCK) receptor antagonist, to digitonin-solubilized CCK receptors from rat pancreas was characterized. [3H](+/-)L-364,718 binding to digitonin-solubilized receptors was assayed using polyethylene glycol precipitation followed by rapid filtration to separate free and bound [3H](+/-)L-364,718. Specific [3H](+/-)L-364,718 binding to solubilized receptors was dependent on the digitonin and receptor concentration and, under optimal conditions, represented greater than 90% of the total binding. Scatchard analysis indicated a single class of binding sites with a Kd of 0.53 nM and a Bmax of 3.1 pmol/mg protein. Specific [3H](+/-)L-364,718 binding to solubilized CCK receptors was inhibited by both CCK receptor agonists and antagonists in a stereospecific manner. After solubilization, the affinities of various antagonists to displace specific [3H](+/-)L-364,718 binding were similar to those obtained with membrane-bound receptors; however, the affinities of CCK agonists were reduced 10-100 times. Collectively, the data presented indicate that [3H](+/-)L-364,718 represents a new antagonist ligand which has apparent advantages over the agonist ligand [125I]CCK in assaying digitonin-solubilized receptors. Gel filtration of the digitonin-solubilized CCK receptors followed by [3H](+/-)L-364,718 binding determinations revealed an estimated molecular weight of 400,000 daltons.     

Characterization of the binding of [3H]L-158,809: a new potent and selective nonpeptide angiotensin II receptor (AT1) antagonist radioligand.

[3H]L-158,809, a new potent and AT1-selective nonpeptide angiotensin II receptor antagonist, bound saturably and reversibly to rat adrenal membranes. Scatchard and Hill plot analyses indicated a single class of high affinity (Kd = 0.66 nM) binding sites. The relative potencies of various angiotensin II-related peptide and nonpeptide antagonists in displacing [3H]L-158,809 binding correlated with their potencies in displacing the binding of 125I-Sar1,Ile8-angiotensin II to adrenal AT1 receptors. [3H]L-158,809 binding to adrenal membranes was not affected by addition of guanosine-5'-(beta,gamma-imido)triphosphate or various pharmacological agents known to interact with other common peptide and nonpeptide receptor systems. The potencies of angiotensin II receptor agonists, but not antagonists, in inhibiting specific [3H]L-158,809 binding were decreased in the presence of guanosine-5'-(beta,gamma-imido)triphosphate. Specific [3H]L-158,809 binding was also observed in rat liver and kidney. Collectively, the data indicate that [3H]L-158,809 represents a new, potent, nonpeptide, antagonist radioligand suitable for the study of angiotensin II AT1 receptors.     

Hybrid cholecystokinin-A antagonists based on molecular modeling of lorglumide and L-364,718.

A series of novel nonpeptide cholecystokinin-A (CCK-A) antagonists have been synthesized. Designed on the basis of the structural homology between lorglumide and L-364,718, as investigated with molecular modeling, these compounds constitute a link between the N-acylglutamic acid and 3-amino-5-phenyl-1,4-benzodiazepin-2-one derived antagonists. The prepared compounds were tested in vitro as antagonists of the binding of [3H]-(+/-)-L-364,718 and [3H]-CCK-8(S) to rat pancreas and guinea pig brain membranes, respectively. All compounds proved to be selective for the (peripheral) CCK-A receptor, the most potent analogue, 6, having a Ki value of 90 nM. The structure-activity profile of the series of hybrid compounds relates closest to that of the N-acylglutamic acid derived antagonists.     

Comparison of CCK-8 receptors in the pancreas and brain of rats using CCK-8 analogues

The characteristics of cholecystokinin (CCK) receptors in rat pancreatic acini and in various regions of the brain were examined using synthetic CCK-8 or CCK-7 analogues. 3H-propionylated CCK-8 [( 3H]CCK-8) was used as a ligand. 1) The pancreatic CCK receptor had a single high affinity binding component with a dissociation constant, Kd, of 0.76 nM and a maximum number of specific binding sites, Bmax, of 271.91 fmol/mg protein. On the other hand, the CCK receptor in the cerebral cortex had a Kd of 1.66 nM and a Bmax of 30.15 fmol/mg protein. 2) The order of the potencies of CCK-7 and CCK-8 analogues with a substitution at position 3 or 4 to displace [3H]CCK-8 specific binding to the pancreatic acini was as follows: CCK-8 greater than CCK-7 = SucCCK-7 greater than Suc[Sar3]CCK-7 greater than Suc[D-Trp3]CCK-7 greater than Suc[D-Ala3]CCK-7 greater than [D-Trp4]CCK-8 = [D-Ala4] CCK-8. This order of potencies of CCK analogues was greatly different from that in the cerebral cortex. 3) The carboxy-terminal tetra-peptide (CCK-4) and penta-peptide (CCK-5) had very weak potencies in displacing [3H]CCK-8 binding in the pancreatic acini, which were 20 to 30-fold less than their potencies in the cerebral cortex. These results suggest that the recognition sites for CCK analogues in the pancreatic and brain CCK receptors are different.     

Characterization of two novel cholecystokinin tetrapeptide (30-33) analogues, A-71623 and A-70874, that exhibit high potency and selectivity for cholecystokinin-A receptors.

Based on their relative affinities for cholecystokinin octapeptide (26-33) (CCK-8), cholecystokinin tetrapeptide (30-33) (CCK-4), desulfated CCK-8, and gastrin, cholecystokinin (CCK) receptors have been classified as CCK-A (alimentary) and CCK-B (brain). Selective nonpeptide antagonists of CCK-A and CCK-B receptors, as well as highly selective CCK-A and CCK-B peptide agonists, have been described. We report here the characterization of two novel CCK-4-based peptides, A-71623 and A-70874. In radioligand binding assays, the IC50 values for A-71623 and A-70874 were 3.7 and 4.9 nM in guinea pig pancreas (CCK-A) and 4500 and 710 nM in cerebral cortex (CCK-B), respectively. Both were agonists in stimulating pancreatic amylase release, and their stimulatory effects were potently inhibited by the CCK-A antagonist L-364,718. A-71623 was a full agonist and A-70874 was a partial agonist (approximately 80%) in stimulating phosphoinositide breakdown in pancreas. Both peptides also were potent agonists in stimulating CCK-A receptors in the ileum. They were, however, weak and behaved as partial agonists in calcium studies in NCI-H345 cells, which possess CCK-B/gastrin receptors. In guinea pig gastric glands, the affinities of A-71623 and A-70874 for the CCK-B/gastrin receptor were 11 and 1.6 microM, respectively. These results demonstrate that A-71623 and A-70874 are potent and selective agonists at CCK-A receptors. The preferential interaction of these novel CCK-4 analogs with CCK-A receptors is in contrast to other CCK-4-based peptides, which are primarily selective for CCK-B receptors. In addition, A-71623 and A-70874 are the first two examples of potent CCK-A agonists that do not contain a tyrosine residue whose sulfation is required for potent CCK-A agonist activity of larger peptides.     

Characterization of histamine type 1 receptors on natural suppressor lymphoid cells

Using the radioligand H1 antagonist [3H]pyrilamine, we have characterized the histamine type 1 receptor on cloned murine natural suppressor cells (NS). A single, specific binding site for [3H]pyrilamine exists on these cells. The binding was saturable and reversible by various specific H1 receptor antagonists. The rank order of potency for displacement of [3H]pyrilamine binding from the H1 receptor by H1 receptor antagonists was promethazine = pyrobutamine greater than pyrilamine greater than diphenhydramine greater than chlorpheniramine. The histamine type-2 agonists, impromidine and dimaprit, and antagonists, cimetidine and ranitidine, as well as selected non-histamine agonists, did not displace [3H]pyrilamine from its binding sites on the natural suppressor cells. The data indicate that the theoretical KD was 1.1 +/- 0.3 X 10(-7) M while the measured KD of binding for [3H]pyrilamine was 6.0 +/- 0.8 X 10(-8) M; the maximum binding was 4.13 nM and the number of binding sites/cell was 2.14 +/- 0.29 X 10(6) (N = 3).     

A novel effect of rebamipide: generation of [Ca(2+)](i) oscillations through activation of CCK(1) receptors in rat pancreatic acinar cells

The protective effect of 2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl]-propionic acid (rebamipide) on gastric mucosa is well established. Here we demonstrate that rebamipide acts on pancreatic acinar cells to generate oscillations of intracellular Ca(2+) concentration ([Ca(2+)](i)) through the activation of cholecystokinin subtype 1 (CCK(1)) receptors. At concentrations higher than 5 microM, rebamipide induced [Ca(2+)](i) oscillations in individual fura-2-loaded pancreatic acinar cells. The frequency of oscillations increased with increasing concentrations of rebamipide, while the latency between stimulation of cells and initiation of [Ca(2+)](i) oscillations decreased with increasing concentration. The [Ca(2+)](i) oscillations evoked by rebamipide were inhibited by the CCK(1) receptor antagonist L-364,718 but not by atropine or the CCK(2) receptor antagonist L-365,260 indicating that rebamipide is a nonpeptide CCK(1) receptor agonist.     

Demonstration of α-adrenoceptors in the rabbit heart by [3H]-dihydroergocryptine binding

For direct identification of alpha-adrenoceptors in a membrane fraction of the rabbit heart the potent alpha-adrenoceptor antagonist [3H]-dihydroergocryptine ([3H]-DHE) was used. 1. The binding of [3H]-DHE was saturable with 80 fmol of [3H]-DHE bound/mg protein and of high affinity with an equilibrium dissociation constant (KD) of 11.5 nM. Binding of [3H]-DHE (6 nM) was rapid (t 1/2 = 2 min) and readily reversible. From the ratio of the rate constants for forward (K1 = 1.97 X 10(7) M-1 min-1) and reverse (K2 = 0.206 min-1) reactions a KD-value of 10 nM was calculated, which is in good agreement with that obtained by equilibrium studies. 2. Adrenergic agonists compete for [3H]-DHE binding in an order to potency: (-)adrenaline greater than (-)phenyleprine greater than (-)isoprenaline and adrenergic antagonists in the order: phentolamine greater than yohimbine greater than (-)propranolol. Binding is stereospecific as indicated by the greater potency of (-)adrenaline than (+/-)adrenaline in displacing [3H]-DHE from the binding sites. 3. For comparison the binding of the potent beta-adrenoceptor antagonist (-)[3H]-dihydroalprenolol ((-)[3H]-DHA) was measured in the same membrane fraction. The number and affinity of beta-adrenoceptors amounted to 115 fmol of (-)[3H]-DHA bound/mg protein at saturation and KD = 7.9 nM. Adrenergic agonists compete for (-)[3H]-DHA binding in an order of potency: (-)isoprenaline greater than (-)adrenaline greater than (-)phenylephrine; and adrenergic antagonists in the order: (-)prapranolol greater than phentolamine. 4. It is concluded that in a membrane fraction of the rabbit heart there exist binding sites for [3H]-DHE which have characteristics indistinguishable from alpha-adrenoceptors. Thus the present results are in agreement with previously reported data on the existence of cardiac alpha-adrenoceptors in the rabbit heart (Schümann et al., 1974; Endoh et al., 1976b).     

Boc-Trp-Orn(Z)-Asp-NH2 and derivatives: a new family of CCK antagonists.

The respective roles of the benzyloxycarbonyl group (Z) and of the N-terminal tripeptide moiety in the antagonist properties of the cholecystokinin CCK8 analogue Boc-[Nle28,Orn(Z)31]CCK27-33 (Marseigne et al. J. Med. Chem. 1988, 31, 966.) were studied with the following derivatives: Boc-[Nle28,Orn(X)31]CCK27-33, Boc[Nle28,Orn(X)31]CCK27-32, Boc-[Orn(X)31]CCK30-33, and Boc-[Orn(X)31]CCK30-32 (X = Z, Boc, H). These derivatives, the synthesis of eight of which is reported here, were tested for their abilities to inhibit the binding of [3H]pCCK8 to guinea pig pancreatic and brain membranes and for their potencies in stimulating amylase release from guinea pig pancreatic acini. None of the Z derivatives produced amylase secretion, but they competitively antagonized the stimulation induced by CCK8. The deletion of the N-terminal tripeptide and/or Phe-NH2(33) residue did not play a key role in the recognition of peripheral receptors and in the activity of these peptides, whereas replacement of the Z group by a Boc group slightly decreased the affinities of the compounds for both pancreatic and brain binding sites and their potencies as peripheral antagonists. Moreover, the tetrapeptide Boc-Trp-Orn(Boc)-Asp-Phe-NH2 behaved as a partial agonist and analogues in which the Z or Boc groups on the ornithine residue were removed were full agonists. Interestingly, the short peptide derivative Boc-Trp-Orn(Z)-Asp-NH2 displayed the same affinity (KI = 2.0 +/- 0.2 x 10(-7] and the same antagonist activity (pA2 = 6.63) as its parent compound Boc-[Nle28,Orn(Z)31]CCK27-33. This tripeptide could be an interesting tool for studying the structural relationships between peptide and non-peptide CCK antagonists.     

Mechanism of corticotropin-releasing factor type I receptor regulation by nonpeptide antagonists

Mechanisms of nonpeptide ligand action at family B G protein-coupled receptors are largely unexplored. Here, we evaluated corticotropin-releasing factor 1 (CRF(1)) receptor regulation by nonpeptide antagonists. The antagonist mechanism was investigated at the G protein-coupled (RG) and uncoupled (R) states of the receptor in membranes from Ltk(-) cells expressing the cloned human CRF(1) receptor. R was detected with the antagonist (125)I-astressin with 30 microM guanosine 5'-O-(3-thiotriphosphate present, and RG detected using (125)I-sauvagine. At the R state, nonpeptide antagonists antalarmin, NBI 27914, NBI 35965, and DMP-696 only partially inhibited (125)I-astressin binding (22-32% maximal inhibition). NBI 35965 accelerated (125)I-astressin dissociation and only partially increased the IC(50) value of unlabeled sauvagine, CRF, and urocortin for displacing (125)I-astressin binding (by 4.0-7.1-fold). Reciprocal effects at the R state were demonstrated using [(3)H]NBI 35965: agonist peptides only partially inhibited binding (by 13-40%) and accelerated [(3)H]NBI 35965 dissociation. These data are quantitatively consistent with nonpeptide antagonist and peptide ligand binding spatially distinct sites, with mutual, weak negative cooperativity (allosteric inhibition) between their binding. At the RG state the compounds near fully inhibited (125)I-sauvagine binding at low radioligand concentrations (79-94 pM). NBI 35965 did not completely inhibit (125)I-sauvagine binding at high radioligand concentrations (82 +/- 1%, 1.3-2.1 nM) and slowed dissociation of (125)I-sauvagine and (125)I-CRF. The antagonist effect at RG is consistent with either strong allosteric inhibition or competitive inhibition at one of the peptide agonist binding sites. These findings demonstrate a novel effect of R-G interaction on the inhibitory activity of nonpeptide antagonists: Although the compounds are weak inhibitors of peptide binding to the R state, they strongly inhibit peptide agonist binding to RG. Strong inhibition at RG explains the antagonist properties of the compounds.     

Characterization of neuronal cholecystokinin receptor by L-364,718 in Auerbach's plexus

The release of [3H]acetylcholine [( 3H]ACh) from Auerbach's plexus and the contraction of longitudinal muscle strips in response to the administration of cholecystokinin (CCK) were measured and recorded simultaneously. The peripheral CCK receptor antagonist, 3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl)-1H- indole-2-carboxamide (L-364,718), antagonized the ACh releasing effect of CCK in a dose-dependent manner. The IC50 value and the dissociation constant (KD) were 41.0 +/- 2.0 pM and 0.06 +/- 0.01 nM, respectively. These results suggest that L-364,718 is a very potent antagonist of the neuronal CCK receptors.     

Differential modulation of the associated glycine recognition site by competitive N-methyl-D-aspartate receptor antagonists

The competitive N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonopentanoate and two other five-atom linkage (C-5) omega-phosphono-alpha-amino acid analogs reduced [3H]glycine binding, in a dose-dependent manner, to a maximum of 45-55%, whereas seven-atom linkage (C-7) analogs had significantly less effect. The IC50 of the C-5 antagonists for the inhibition of [3H]glycine binding closely paralleled their potency both in displacing NMDA-selective L-[3H]glutamate binding and in negatively modulating (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne maleate ([3H]MK-801) binding. Additionally, reduction of glycine binding by the C-5 antagonists was reversed by both NMDA receptor agonists and C-7 competitive NMDA antagonists, providing evidence that the site of action of these C-5 antagonists is the NMDA recognition site, resulting in indirect modulation of the glycine site. These data imply a functional coupling between the NMDA and associated glycine recognition sites and, furthermore, suggest a differential interaction of C-5 and C-7 competitive NMDA antagonists with the NMDA receptor complex.     

3H]RX821002: a new tool for the identification of alpha 2A-adrenoceptors

The human adenocarcinoma cell-line HT29 was used as a model to investigate the binding properties of a new antagonist radioligand of the imidazoline series, [3H]RX821002. All aspects of [3H]RX821002 binding conclusively prove that this radioligand is a valuable tool for labelling alpha 2A-adrenoceptors. [3H]RX821002 binding was very rapid and reversible. Computer-assisted analysis of kinetic data revealed association and dissociation time courses consistent with a simple bimolecular reaction. Saturation isotherms indicated that [3H]RX821002 labeled with high affinity a single population of non-interacting sites displaying a KD of 1.7 +/- 0.1 nM. Adrenoceptor agonists and antagonists inhibited [3H]RX821002 and [3H]yohimbine binding with a strictly similar rank order of potency which is characteristic of alpha 2A-adrenoceptors. The binding parameters of [3H]RX821002 were compared with those of other commercially available [3H]antagonists, [3H]yohimbine and [3H]idazoxan. Analysis of the saturation isotherms for the three radioligands showed that (1) [3H]RX821002 was the radioligand exhibiting the lower percentage of non-specific binding and the better affinity, (2) the Bmax of [3H]RX821002 was significantly higher than that of [3H]yohimbine. The difference in Bmax was not due to better labelling of one of the two affinity states of the receptor but was greatly reduced in glycylglycine buffer, suggesting that, in Tris-Mg2+ buffer, [3H]yohimbine does not label the entire alpha 2-adrenoceptor population.     

Thermodynamic analysis does not allow discrimination of agonists and antagonists at human CCK2S-receptors

Studies have shown that measurement of thermodynamic parameters (enthalpy, DeltaH degrees and entropy, DeltaS degrees ) can allow discrimination of agonists and antagonists (e.g. Weiland, G.A., Minneman, K.P., Molinoff, P.B., 1979. Fundamental difference between the molecular interactions of agonists and antagonists with the beta-adrenergic receptor. Nature, 281, 114.). Recently, we found that agonists and antagonists were not thermodynamically-distinguished at cholecystokinin (CCK)2-receptors in rat cerebral cortex. However, in this study, the possibility that thermodynamic discrimination at CCK2-receptors exists but that it was not detected, could not be excluded because radioligand binding studies and functional assays were performed in different rat tissues. Therefore, we have repeated these studies using the recombinant CCK2 short isoform (CCK2S)-receptor expressed in NIH3T3 cells, so that ligand affinity (pKI) and intrinsic activity (alpha) measurements could be made in exactly the same receptor system. CCK-8S but not R-L-365,260, S-L-365,260, JB95008, JB93242 or PD134,308 expressed intrinsic activity in an IP assay. The pKD of [3H]-JB93182 decreased with increasing temperature. pKI values for antagonists (R-L-365,260, S-L-365,260, JB95008) and agonists (pentagastrin, CCK-8S) were higher at 4 than at 30 degrees C. There was no effect of temperature on pKI values for the antagonists, PD134,308 and JB93242. Therefore, CCK2-receptor agonists and antagonists at human CCK2S-receptors cannot be discriminated by thermodynamic analysis.     

"Gastrin" and "CCK" receptors on histamine- and somatostatin-containing cells from rabbit fundic mucosa-II. Characterization by means of selective antagonists (L-364,718 and L-365,260).

In the preceding paper, by means of selective agonists to gastrin (HG-17) and cholecystokinin (CCK-39), we evidenced the existence of "gastrin-type" receptors that could regulate histamine release and "CCK-type" receptors that could stimulate somatostatin release in isolated rabbit fundic non-parietal cells (F1 cells). Furthermore, these receptors could induce phosphoinositide breakdown. To confirm the involvement of these receptor types in these biological and biochemical processes, we used selective antagonists, L-364,718 (3-(benzoylamino)-benzodiazepine) specific to "CCK-A-type" receptor and L-365,260 (3-(acylamino)-benzodiazepine) specific to "gastrin/CCK-B-type" receptor. Neither L-364,718 nor L-365,260 alone caused any significant stimulation of [3H]inositol phosphate ([3H]InsP) production and release of histamine or somatostatin-like immunoreactivity (SLI). Each analogue inhibited in a dose-dependent manner [125I]HG-17 or [125I]CCK-39 binding to F1 cells, [3H]InsP accumulation and histamine and SLI release stimulated by HG-17 or CCK-39. L-365,260 appeared to be 30-70 times more potent than L-364,718 in inhibiting [125I]HG-17 binding to F1 cells, as well as HG-17-induced [3H]InsP accumulation and HG-17-or CCK-39-enhanced histamine release (IC50 values: approximately 5-20 nM for L-365,260 and approximately 200-1500 nM for L-364,718). In contrast, L-364,718 was 200 to 400 times more potent than L-365,260 in inhibiting [125I]CCK-39 binding to F1 cells, CCK-39-induced [3H]-InsP accumulation and SLI release stimulated by CCK-39 or HG-17 (IC50 values: approximately 0.3-1 nM for L-364,718 and 100-200 nM for L-365,260). These results led to conclude: (i) the existence of a "gastrin-type" receptor related to histamine release: (ii) the existence of a "CCK-A-type" receptor related to somatostatin release; (iii) the existence of "gastrin type" and "CCK-A-type" receptors linked to the phosphoinositide breakdown pathway.     

In vivo binding affinities of cholecystokinin agonists and antagonists determined using the selective CCKB agonist [3H]pBC 264.

The respective role of central vs. peripheral CCK-B receptors in the recently reported anxiolytic effects of CCK-B antagonists remains to be firmly established. We therefore investigated the in vivo binding properties of cerebral CCK receptors after i.c.v. injection into mice of [3H]pBC 264 ([3H]propionyl-Tyr(SO3H)-gNle-mGly- Trp-(NMe)Nle-Asp-Phe-NH2), a highly potent, peptidase-resistant and selective CCK-B agonist. The specific binding of [3H]pBC 264 was reversible and saturable. The dose producing 50% receptor occupancy was 25 pmol and the Bmax was 0.9 pmol/brain 15 min after injection. I.c.v. administered CCK8 (ID50 8500 pmol) was 200-fold less potent than pBC 264 (ID50 43 pmol) in inhibiting specific [3H]pBC 264 binding; CCK8NS, CCK5 and CCK4 being slightly less potent than CCK8. Aminopeptidases play a major role in degrading CCK8 since the protected analog pCCK8 or CCK8 in the presence of an aminopeptidase inhibitor exhibited higher affinities than CCK8. I.v. administration of pBC 264 (20 mg/kg) inhibited [3H]pBC 264 specific binding by about 72%, confirming its ability to enter the brain. In contrast, CCK4 was unable to modify [3H]pBC 264 binding. As expected, the CCK-A antagonist (L364,718) did not inhibit [3H]pBC 264 binding, while at the highest dose used (40 mg/kg i.p.) the CCK-B antagonist (L365,260) inhibited binding by 20%. Several hypotheses are discussed to account for the very low i.v. doses of CCK4 and L365,260 needed to produce anxiogenic and anxiolytic responses, respectively.     

Pharmacologic characterization of the oxytocin receptor in human uterine smooth muscle cells

[(3)H]-oxytocin was used to characterize the oxytocin receptor found in human uterine smooth muscle cells (USMC). Specific binding of [(3)H]-oxytocin to USMC plasma membranes was dependent upon time, temperature and membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high-affinity binding sites with an apparent equilibrium dissociation constant (K(d)) of 0.76 nM and a maximum receptor density (B(max)) of 153 fmol mg(-1) protein. The Hill coefficient (n(H)) did not differ significantly from unity, suggesting binding to homogenous, non-interacting receptor populations. Competitive inhibition of [(3)H]-oxytocin binding showed that oxytocin and vasopressin (AVP) receptor agonists and antagonists displaced [(3)H]-oxytocin in a concentration-dependent manner. The order of potencies for peptide agonists and antagonists was: oxytocin>[Asu(1,6)]-oxytocin>AVP= atosiban>d(CH(2))(5)Tyr(Me)AVP>[Thr(4),Gly(7)]-oxytocin>dDAVP, and for nonpeptide antagonists was: L-371257>YM087>SR 49059>OPC-21268>SR 121463A>OPC-31260. Oxytocin significantly induced concentration-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) and hyperplasia in USMC. The oxytocin receptor antagonists, atosiban and L-371257, potently and concentration-dependently inhibited oxytocin-induced [Ca(2+)](i) increase and hyperplasia. In contrast, the V(1A) receptor selective antagonist, SR 49059, and the V(2) receptor selective antagonist, SR 121463A, did not potently inhibit oxytocin-induced [Ca(2+)](i) increase and hyperplasia. The potency order of antagonists in inhibiting oxytocin-induced [Ca(2+)](i) increase and hyperplasia was similar to that observed in radioligand binding assays. In conclusion, these data provide evidence that the high-affinity [(3)H]-oxytocin binding site found in human USMC is a functional oxytocin receptor coupled to [Ca(2+)](i) increase and cell growth. Thus human USMC may prove to be a valuable tool in further investigation of the physiologic and pathophysiologic roles of oxytocin in the uterus. British Journal of Pharmacology (2000) 129, 131 - 139     

2-Naphthalenesulphonyl L-aspartyl-(2-phenethyl)amide (2-NAP)--a selective cholecystokinin CCKA-receptor antagonist.

1. The in vitro pharmacological characterization of the sodium salt of 2-naphthalenesulphonyl 1-aspartyl-(2-phenethyl)amide [2-NAP], a hydrophilic compound derived from the C-terminal aspartate-phenylalanine dipeptide of cholecystokinin (CCK), is described. 2. 2-NAP behaved as a competitive antagonist of sulphated cholecystokinin octapeptide (CCK-8) at CCKA-receptors in both intact tissue bioassays (guinea-pig gall bladder, pancreas and ileum, human and rabbit gall bladder) and a radioligand displacement assay (guinea-pig pancreatic cells). The mean pKB, over assays, was 6.5. 3. Compared to the other assays, the rabbit gall bladder assay gave a significantly higher pKB estimate [7.0] for 2-NAP and a significantly lower estimate [8.9] for devazepide (formerly L-364,718 and MK-329), a well-characterized CCKA-receptor antagonist; these anomalous results suggest that a different class of CCKA-receptors may be involved. 4. 2-NAP, was found to be highly selective, having at least 300 fold greater affinity for CCKA-receptors than for 50 other pharmacological loci, including gastrin/CCKB, as estimated by bioassay or radioligand displacement.     

Binding of [3H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes--a new, selective antagonist radioligand for A(2A) adenosine receptors.

The present study describes the preparation and binding properties of a new, potent, and selective A_2A adenosine receptor (AR) antagonist radioligand, [³H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine ([³H]MSX-2). [³H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [³H]MSX-2 labeled a single class of binding sites with high affinity (K_d=8.0 nM) and limited capacity (B_max=1.16 fmol·mg⁻¹ of protein). The presence of 100 μM GTP, or 10 mM magnesium chloride, respectively, had no effect on [³H]MSX-2 binding. AR agonists competed with the binding of 1 nM [³H]MSX-2 with the following order of potency: 5′-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine (CGS-21680)>2-chloroadenosine (2-CADO)>N⁶-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3,7-dimethyl-1-propargylxanthine (BS-DMPX)>1,3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5,6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2,3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K_i values for antagonists were in accordance with data from binding studies with the agonist radioligand [³H]CGS21680, while agonist affinities were 3–7-fold lower. [³H]MSX-2 is a highly selective A_2A AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20–30%, at 1 nM.