Selectivity of oxomemazine for the M1 muscarinic receptors

The binding characteristics of pirenzepine and oxomemazine to muscarinic receptor were studied to evaluate the selectivity of oxomemazine for the muscarinic receptor subtypes in rat cerebral microsomes. Equilibrium dissociation constant (KD) of (-)-[3H]quinuclidinyl benzilate([3H]QNB) determined from saturation isotherms was 64 pM. Analysis of the pirenzepine inhibition curve of [3H]QNB binding to cerebral microsome indicated the presence of two receptor subtypes with high (Ki = 16 nM, M1 receptor) and low (Ki = 400 nM, M3 receptor) affinity for pirenzepine. Oxomemazine also identified two receptor subtypes with about 20-fold difference in the affinity for high (Ki = 84 nM, OH receptor) and low (Ki = 1.65 microM, OL receptor) affinity sites. The percentage populations of M1 and M3 receptors to the total receptors were 61:39, and those of OH and OL receptors 39:61, respectively. Both pirenzepine and oxomemazine increased the KD value for [3H]QNB without affecting the binding site concentrations and Hill coefficient for the [3H]QNB binding. Oxomemazine had a 10-fold higher affinity at M1 receptors than at M3 receptors, and pirenzepine a 8-fold higher affinity at OH receptors than at OL receptors. Analysis of the shallow competition binding curves of oxomemazine for M1 receptors and pirenzepine for OL receptors yielded that 69% of M1 receptors were of OH receptors and the remaining 31% of OL receptors, and that 29% of OL receptors were of M1 receptors and 71% of M3 receptors. However, M3 for oxomemazine and OH for pirenzepine were composed of a uniform population. These results suggest that oxomemazine could be classified as a selective drug for M1 receptors and also demonstrate that rat cerebral microsomes contain three different subtypes of M1, M3 and the other site which is different from M1, M2 and M3 receptors.     

Antimuscarinic effects of antihistamines: quantitative evaluation by receptor-binding assay

Quantitative evaluation of antimuscarinic effects of antihistamines (H1- and H2-receptor antagonists) was carried out using a receptor-binding assay. The -inhibition constants (Ki values) of twenty seven H1-receptor antagonists, one related antidepressant and three H2-receptor antagonists at H1-receptors and muscarinic receptors in the bovine cerebral cortex were determined. All the H2-receptor antagonists examined showed very low affinity for the muscarinic receptors. On the other hand, some H1-receptor antagonists (mequitazine, cyproheptazine, clemastine, diphenylpyraline, promethazine, homochlorcyclizine and alimemazine) had high affinity for the muscarinic receptors (Ki = 5.0-38 nM). Another group of H1-receptor antagonists (mepyramine, terfenadine, metapyrilen, azelastine, hydroxyzine and meclizine) had low affinity for the muscarinic receptors (Ki = 3,600-30,000 nM). Thus, a broad range of antimuscarinic potencies among the antihistamines was demonstrated. These results should provide helpful information with regard to the clinical and experimental use of antihistamines.     

Synthesis and dopamine receptor affinities of N-alkyl-11-hydroxy-2-methoxynoraporphines: N-alkyl substituents determine D1 versus D2 receptor selectivity

We developed a procedure to synthesize a series of N-alkyl-2-methoxy-11-hydroxynoraporphines from thebaine and evaluated their binding affinities at dopamine D1 and D2 receptors in rat forebrain tissue. At D2 receptors, the most potent 10,11-catechol-aporphine was (R)-(-)-2-methoxy-N-n-propylnorapomorphine (D2, Ki = 1.3 nM; D1, Ki = 6450 nM), and the most selective and potent 11-monohydroxy aporphine was (R)-(-)-2-methoxy-11-hydroxy-N-n-propylnoraporphine (D2, Ki = 44 nM; D1, Ki = 1690 nM). In contrast, the N-methyl congeners (R)-(-)-2-methoxy-11-hydroxy-N-methyl-aporphine (D1 vs D2, Ki = 46 vs 235 nM) showed higher D1 than D2 affinity, indicating that N-alkyl substituents have major effects on D2 affinity and D2/D1 selectivity in such 2-methoxy-11-monohydroxy-substituted aporphines.     

7-Deaza-2-phenyladenines: structure-activity relationships of potent A1 selective adenosine receptor antagonists

A series of derivatives of 7-deazapurines with varying substituents in the 2-, 6-, and 9-position was synthesized in an attempt to improve the adenosine receptor affinity and A1 or A2 selectivity. The adenosine receptor affinities were assessed by measuring the inhibition of [3H]-(R)-N6-(phenylisopropyl) adenosine (R-PIA) binding to rat brain A1 and inhibition of [3H]-5'-(N-ethylcarboxamido)adenosine (NECA) binding to rat striatum A2 adenosine receptors. A selected set of compounds representing the main structural variations was further examined in adenosine receptor coupled adenylate cyclase assays. All tested compounds antagonized the inhibition of adenylate cyclase elicited by interaction of R-PIA with A1 receptors in rat fat cell membranes and the activation of adenylate cyclase elicited by interaction of NECA with A2 receptors of pheochromocytoma PC12 cell membranes. The results indicate that 7-deazahypoxanthines have a potential for A2 selectivity, while all 7-deazaadenines are A1 selective. Introduction of a phenyl residue in the 2-position of 7-deazaadenines increases A1 activity tremendously. 2-(p-Chlorophenyl)-7,8-dimethyl-9-phenyl-7-deazaadenine (29) is potent and specific for the A1 receptors of rat brain (Ki = 122 nM), having no affinity for the A2 receptors of rat striatum. The compound has low activity at the A2 receptors of rat PC12 cell membranes where it appears to act as a noncompetitive inhibitor. A 1-phenylethyl substituent at the 9-position was found to be superior to a phenyl residue in terms of A1 affinity. The most potent A1 antagonist in the present series is the highly A1 selective (790-fold) (R)-7,8-dimethyl-2-phenyl-9-(1-phenylethyl)-7-deazaadenine (31, Ki = 4.7 nM), which is 30-35 times more potent at A1 receptors than its S enantiomer. The solubility of six of the potent 7-deaza-2-phenyladenines was determined by means of an A1 binding assay. Chloro substitution of the 2-phenyl ring appeared to improve the solubility as well as the solubility over A1 affinity ratio of 9-phenyl- and 9-(1-phenylethyl)-substituted 7-deazadenines.     

Evaluation of the receptor selectivity of the H3 receptor antagonists, iodophenpropit and thioperamide: an interaction with the 5-HT3 receptor revealed.

1. In the present study we evaluated the receptor selectivity of the potent histamine H3 receptor antagonist, iodophenpropit (IPP) in comparison with the prototype antagonist, thioperamide. 2. IPP proved to be a potent competitive H3 receptor antagonist as measured against (R)-alpha-methylhistamine-induced inhibition of electrically-evoked contractions of the guinea-pig jejunum (pA2 = 9.12 +/- 0.06, Schild slope: 1.0 +/- 0.1, n = 8). In the same assay, thioperamide was slightly less potent (pA2 = 8.9 +/- 0.2). 3. In radioligand binding studies, IPP showed a high affinity for the H3 receptor. Displacement of [125I]-IPP binding to rat cortex membranes by unlabelled IPP resulted in a Ki value of 0.97 +/- 0.06 nM (n = 3). In contrast, IPP showed only a weak affinity for the histamine H1- and H2 receptor. Displacement of [3H]-mepyramine and [125I]-iodoaminopotentidine binding to respectively guinea-pig H1- and human H2 receptors by IPP resulted in Ki values of 1.71 +/- 0.32 microM (n = 3) and 2.28 +/- 0.81 microM (n = 3). For thioperamide the affinities for the H1-, H2- and H3 receptor were respectively > 10 microM, > 10 microM and 4.3 +/- 1.6 nM (n = 7). 4. Testing IPP and thioperamide in 39 different receptor binding assays revealed that IPP showed relatively high affinity for the 5-hydroxytryptamine 5-HT3 receptor (Ki = 11 +/- 1 nM, n = 3), the alpha 2-adrenoceptor (Ki = 120 +/- 5 nM, n = 3) and the sigma receptor (Ki = 170 +/- 70 nM, n = 3). Thioperamide showed relatively high affinity for the 5-HT3 receptor (Ki = 120 +/- 30 nM, n = 3) and the sigma receptor (Ki = 180 +/- 90 nM, n = 3). 5. Due to the low density of histamine H3 receptors in the brain, the interaction of IPP with the 5-HT3-, the alpha 2- and the sigma receptor might interfere with [125I]-IPP binding to rat cortex membranes. Yet, in this preparation [125I]-IPP binding was not influenced by ondansetron, yohimbine or haloperidol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct labeling of rat M3-muscarinic receptors by [3H]4DAMP

The muscarinic receptors of rat submaxillary gland, rat heart and rat cortex were directly labeled using the ligand [3H]4-diphenylacetoxy-N-methyl-piperidine methiodide [( 3H]4DAMP). In the rat submaxillary gland, [3H]4DAMP predominantly bound with high affinity (Kd = 0.2 nM) to a population of binding sites that displayed the pharmacology of the M3 muscarinic receptor subtype. In rat heart, [3H]4DAMP labeled the M2 muscarinic receptor with low affinity (Kd = 4 nM). In rat cortex [3H]4DAMP predominantly bound to a population of sites with high affinity (Kd = 0.2 nM). The pharmacology of these sites was consistent with [3H]4DAMP labeling both M1 and M3 muscarinic receptors present in rat cortex with high affinity. These data indicate that [3H]4DAMP represents a useful ligand for selectively labeling the M1 and M3 muscarinic receptor subtypes.     

M3 muscarinic cholinoceptors are linked to phosphoinositide metabolism in rat cerebellar granule cells.

Primary cultures of rat cerebellar granule cells are shown to possess a high density (283 +/- 48 fmol/mg of protein) of muscarinic receptor sites, defined using N-[3H]methylscopolamine [( 3H]NMS), with a KD of 0.18 +/- 0.01 nM measured after culture in vitro for 7 days. Displacement of specific [3H]NMS binding demonstrated a muscarinic receptor with low affinity for pirenzepine (Ki: 240 nM); further investigation using antagonists, AF-DX 116 and 4-DAMP to discriminate between M2 and M3 receptors respectively, revealed low M2 affinity (Ki: 600 nM) and high M3 affinity (Ki: 2.4 nM), indicative of the M3 receptor subtype. The robust muscarinic receptor stimulation of [3H]inositol phosphate formation, previously observed in these cells, was confirmed. Inhibition of this response followed a similar profile to the binding data, exhibiting weak inhibitory effects for pirenzepine (Ki: 710 nM) and AF-DX 116 (Ki: 5000 nM), but a potent action for 4-DAMP (Ki: 2.4 nM). The opposite profile seen for AF-DX 116 and 4-DAMP is indicative of a M3 receptor subtype expressed on these cells and linked to phosphoinositide hydrolysis. Further studies demonstrated that M3 receptor stimulation caused a rapid, transient increase in the second messenger inositol 1,4,5-trisphosphate, suggesting that potential Ca(2+)-homeostatic and neuromodulatory effects may be mediated by this response.     

BODIPY-conjugated neuropeptide Y ligands: new fluorescent tools to tag Y1, Y2, Y4 and Y5 receptor subtypes

N-terminal labelled fluorescent BODIPY-NPY peptide analogues were tested in Y1, Y2, Y4 and Y5 receptor-binding assays performed in rat brain membrane preparations and HEK293 cells expressing the rat Y1, Y2, Y4 and Y5 receptors. BODIPY TMR/FL-[Leu31, Pro34]NPY/PYY were able to compete for specific [125][Leu31, Pro34]PYY-binding sites with an affinity similar to that observed for the native peptide at the Y1 (Ki=1-6 nM), Y2 (Ki>1000 nM), Y4 (Ki=10 nM) and Y5 (Ki=1-4 nM) receptor subtypes. BODIPY FL-PYY(3-36) was able to compete for specific Y2 (Ki=10 nM) and Y5 (Ki=30 nM) binding sites, but had almost no affinity in Y1 and Y4 assays. BODIPY FL-hPP was able to compete with high affinity (Ki; 1 and 15 nM) only in Y4 and Y5 receptor-binding assays. BODIPY TMR-[cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP and BODIPY TMR-[hPP(1-17), Ala31, Aib32]NPY were potent competitors only on specific Y5-binding sites (Ki=0.1-0.6 nM). As expected, these fluorescent peptides inhibited forskolin-induced cAMP accumulation, demonstrating that they retained their agonist properties. When tested in confocal microscopy imaging, fluorescent Y1 and Y5 agonists internalized in a time-dependent manner in Y1 and Y5 transfected cells, respectively. These results demonstrate that BODIPY-conjugated NPY analogues retain their selectivity, affinity and agonist properties for the Y1, Y2, Y4 and Y5 receptor subtypes, respectively. Thus, they represent novel tools to study and visualize NPY receptors in living cells.     

Biochemical pharmacology of pirenzepine. Similarities with tricyclic antidepressants in antimuscarinic effects only

5,11-Dihydro-11-[(4-methyl-1-piperazinyl) acetyl]-6H-pyrido[2,3-b][-1,4]-benzodiazepin-6-one dihydrochloride (pirenzepine, Gastrozepin) and some tricyclic antidepressant drugs which show a very close relationship concerning the chemical structure were investigated in numerous binding, uptake and enzymatic studies in vitro. With pirenzepine a high affinity binding could be demonstrated only to muscarinic receptors (Ki = 58 nmol/l). In all other studies pirenzepine had a very weak or no effect at all. In contrast, tricyclic antidepressants bound with high but different affinities to various receptors as known from numerous publications. The highest affinities were found with imipramine at the specific imipramine binding sites (Ki = 9.8 nmol/l) and at the alpha 1-receptor (Ki = 39 nmol/l), with desipramine at the muscarinic receptors (Ki = 88 nmol/l), with mianserin at the H1-(Ki = 3.4 nmol/l) and 5HT2-receptors (Ki = 7.3 nmol/l). Moreover, imipramine and desipramine showed their known substantial inhibition of noradrenaline and/or 5-hydroxytryptamine uptake. Thus, a homogeneous affinity or activity profile of the antidepressants studied does not exist. The only common property of pirenzepine and the tricyclic antidepressants was found to be the high affinity binding to the muscarinic receptors which might explain the common antisecretory action of these agents. Because of the unique specificity of pirenzepine lacking all other effects of the tricyclic antidepressants as demonstrated in this study, it is very unlikely that this drug exerts any antidepressant-like central action.     

Muscarinic receptor subtypes in human and rat colon smooth muscle.

Muscarinic receptor subtypes in human and rat colon smooth muscle homogenates were characterized with [3H]N-methylscopolamine ([3H]NMS) by ligand binding studies. [3H]NMS saturation experiments show the existence of a homogeneous population of non-interacting binding sites with similar affinity (KD values of 1.38 +/- 0.20 nM in human colon smooth muscle and 1.48 +/- 0.47 nM in rat colon smooth muscle) and with Hill slopes close to unity in both samples of tissue. However, a significant (P less than 0.01) increase in muscarinic receptor density (Bmax) is found in human colon (29.9 +/- 2.9 fmol/mg protein) compared with rat colon (17.2 +/- 1.5 fmol/mg protein). Inhibition of [3H]NMS binding by non-labelled compounds shows the following order in human colon: atropine greater than AF-DX 116 greater than pirenzepine. Whereas in rat colon the rank order obtained is atropine greater than pirenzepine greater than AF-DX 116. Atropine and pirenzepine bind to a homogeneous population of binding sites, although pirenzepine shows higher affinity to bind to the sites present in rat colon (Ki = 1.08 +/- 0.08 microM) than those in human colon (Ki = 1.74 +/- 0.02 microM) (P less than 0.05). Similarly, IC50 values obtained in AF-DX 116 competition experiments were significantly different (P less than 0.01) in human colon (IC50 = 1.69 +/- 0.37 microM) than in rat colon (IC50 = 3.78 +/- 0.75 microM). Unlike atropine and pirenzepine, the inhibition of [3H]NMS binding by AF-DX 116 did not yield a simple mass-action binding curve (nH less than 1, P less than 0.01) suggesting the presence of more than one subtype of muscarinic receptor in both species. Computer analysis of these curves with a two binding site model suggests the presence of two populations of receptor. The apparent Ki1 value for the high affinity binding site is 0.49 +/- 0.07 microM for human colon smooth muscle and 0.33 +/- 0.05 microM for rat colon smooth muscle. The apparent Ki2 for the low affinity binding site is 8.01 +/- 1.0 microM for human samples and 6.07 +/- 1.1 microM for rat samples. These values are close enough to suggest that the first subtype of muscarinic receptor may be considered cardiac (M2) and the second subtype glandular (M3). The relative densities of the receptor subtypes are significantly different for both species. Human colon samples show the major densities of subtype M2, 22.62 +/- 1.11 fmol/mg protein, this represents 75.66 +/- 3.73% of the total receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inverse agonist activity of pirenzepine at M2 muscarinic acetylcholine receptors

1. The intrinsic properties of muscarinic ligands were studied through their binding properties and their abilities to modulate the GTPase activity of G proteins coupled to muscarinic M2 receptors in pig atrial sarcolemma. 2. Competition binding experiments were performed with [3H]-oxotremorine-M to assess the affinity of receptors coupled to G proteins (R*), with [3H]-N-methylscopolamine ([3H]-NMS) to estimate the affinities of coupled and uncoupled receptors (R*+R) and with [3H]-NMS in the presence of GppNHp to assess the affinity of uncoupled receptors (R). 3. The ranking of Ki values for the agonist carbachol was R*R*+R>R (174, 155, 115 nM), suggesting inverse agonism. 4. The Vmax of the basal high affinity GTPase activity of pig atrial sarcolemma was increased by mastoparan and decreased by GPAnt-2 indicating the relevance of this activity to G proteins coupled to receptors (R*). The K(M) value (0.26-0.33 microM) was not modified by mastoparan or GPAnt-2. 5. Carbachol increased the Vmax of GTP hydrolysis (EC50 8.1+/-0.3 microM), whereas atropine and AF-DX 116, up to 1 mM, did not modify it. Pirenzepine decreased the Vmax of GTP hydrolysis (EC50 77.5+/-10.3 microM). This effect was enhanced when KCI was substituted for NaCl (EC50 11.0+/-0.8 microM) and was antagonized by atropine and AF-DX 116 (IC50 0.91+/-0.71 and 197+/-85 nM). 6. Pirenzepine is proposed as an inverse agonist and atropine and AF-DX 116 as neutral antagonists at the muscarinic M2 receptor.     

Secoverine is a non-selective muscarinic antagonist on rat heart and brain receptors

In an attempt to determine if the selectivity of secoverine observed in vivo and in isolated tissues might be due to selective association with muscarinic receptor subtypes, we analyzed the binding of secoverine to three different receptors with specific radioligands: rat cardiac receptors (M2 receptors with low affinity for atropine), and rat cerebral cortical M1 receptors and M2 receptors with high affinity for atropine. At concentrations up to 10(-6) M, secoverine interaction with muscarinic receptors was competitive and of high affinity (Ki 4.10(-9) M) for cardiac and brain receptors. A detailed analysis using, in addition to [3H]N-methylscopolamine, the agonist [3H]oxotremorine-M (selective for high affinity binding sites at cardiac receptors) and the M1-selective antagonist [3H]pirenzepine at brain receptors, revealed identical affinities towards both receptor types, making it unlikely that secoverine distinguished the different muscarinic receptor subtypes. At concentrations between 10(-6) and 10(-3) M, secoverine interaction with an additional receptor site resulted in profound changes of tracer kinetics, suggesting the formation of a ternary complex (secoverine-radioligand-muscarinic receptor). The potency of secoverine for provoking this allosteric interaction was both tracer- and tissue-dependent. It is concluded that secoverine does not differentiate between M1, brain M2 and cardiac M2 receptors or between cardiac receptors with high, low and very low affinity for agonists. At very high concentrations secoverine recognized an allosteric site on the muscarinic receptors and reduced the dissociation rates of the 3H-ligands.     

The cholinergic pharmacology of tetrahydroaminoacridine in vivo and in vitro.

1. The effect of tetrahydroaminoacridine (THA) on cholinergically mediated behaviour in the rat and mouse has been investigated. In addition the actions of this compound on cholinesterase activity and on muscarinic and nicotinic receptors has also been examined. 2. Administration of THA (5-20 mg kg-1, i.p.) produced a dose-dependent increase in tremor, hypothermia and salivation in both rats and mice. A similar profile of activity was seen following physostigmine (0.1-0.6 mg kg-1) administration. 3. THA was approximately fifty fold less potent than physostigmine in inducing behavioural change but its effects persisted for over twice as long as those of physostigmine. For example THA-induced hypothermia was still present at 4 h in the mouse and 8 h in the rat. 4. In vitro THA was a potent non-competitive inhibitor of rat brain cholinesterase (IC50: 57 +/- 6 nM) and bovine erythrocyte acetylcholinesterase (IC50: 50 +/- 10 nM) but was a more potent inhibitor of horse serum butyrylcholinesterase (IC50: 7.2 +/- 1.4 nM). 5. Radioligand binding studies indicated that THA binds non-selectively but with moderate potency to both M1 (Ki: 600 nM) and M2 (Ki: 880 nM) muscarinic receptors. THA also interacted with the allosteric site present on cardiac M2 receptors. 6. It is concluded that THA is a reversible non-competitive inhibitor of cholinesterase with a long half life (compared with physostigmine). It also may antagonize muscarinic receptors at high doses. The long half life may account for its reported efficacy in the treatment of Alzheimer's disease.     

Effect of steroid hormones on muscarinic receptors of bronchial smooth muscle.

1. Glucocorticosteroid may relieve bronchospasm by mediating changes in the muscarinic receptor concentration and/or its affinity. 2. Cholinergic muscarinic receptors were determined by using Scatchard's plots from radioligand binding assays of 0.13-3.2 nM [3H]quinuclidinyl benzylate binding to the membrane fraction of bronchial smooth muscle (BSM). 3. The concentration of muscarinic receptor in BSM of normal rat was 57 +/- 3 fmol mg protein and the dissociation constant was 0.07 +/- 0.02 nM. Dexamethasone and corticosterone reduced muscarinic receptor concentration to 50-60% of basal with no changes in receptor affinity. No changes were found in rat treated with deoxycorticosterone. 4. These findings suggest that glucocorticoids but not mineralocorticoid relieve bronchospasm at least partly by reducing the cholinergic hypersensitivity.     

Interaction of DNA-intercalating antitumor agents with adrenoceptors

The interaction between some examples of mononuclear and binuclear DNA-intercalating antitumor agents and alpha- and beta-adrenoceptors has been studied using radioligand-binding assays. Competition for 125I-BE 2254, [3H]rauwolscine, and (-)-[3H]dihydroalprenolol binding was used to assess affinity for alpha 1-, alpha 2-, and beta-adrenoceptor-binding sites, respectively. Two homologous series of alkyl-linked diacridines and diquinolines were found to interact poorly with beta-adrenoceptors, with only the largest members having appreciable affinity. By contrast, these compounds bind strongly and in a complex manner to alpha 1- and alpha 2-adrenoceptors. The affinity of diacridines for both alpha-adrenoceptor classes has a parabolic dependence on alkyl chain length with the hexyl and pentyl derivatives being the most potent at the alpha 1- (Ki = 11.5 +/- 2.3 nM) and alpha 2- (Ki = 143 +/- 26 nM) binding sites, respectively. The dependence of inhibition constants on linker chain length for the diquinolines is more complicated, with the ethyl- and heptyl-linked dimers having the greatest affinity for each alpha subclass. There is a nadir in affinity for the pentyl and butyl ligands and an increase in dissociation constant for octyl and longer homologues. Thus, the ethyl diquinoline has Ki values of 6.6 +/- 1.2 and 110 +/- 14 nM for the alpha 1- and alpha 2- adrenoceptors, respectively, and, correspondingly, the heptyl derivative has values of 39 +/- 4 and 51 +/- 1 nM. These findings are discussed with respect to a model of the alpha-adrenoceptor in which the radioligand-binding site is situated in a trench or cleft, surrounded by a flat surface bounded by walls. Daunomycin was found to have no affinity for adrenoceptors of any type and mitoxantrone similarly fails to interact with alpha 2- and beta-adrenoceptors, but binds to the alpha 1 subclass with an inhibition constant (Ki) of 3930 +/- 420 nM. Bisantrene also has no affinity for beta-adrenoceptors but binds to alpha 1- and alpha 2- adrenoceptors with Ki values of 145 +/- 24 and 2310 +/- 430 nM, respectively. Among the mononuclear acridine drugs studied, only nitracrine shows detectable interaction with beta-adrenoceptors (Ki = 760 +/- 50 nM). This compound, like bisantrene, has high affinity for the alpha 1-adrenoceptor (Ki = 131 +/- 17 nM) and moderate affinity for the alpha 2 subclass (Ki = 2180 +/- 500 nM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and characterization of a high affinity radioiodinated probe for the alpha 2-adrenergic receptor

The availability of radioiodinated probes has facilitated the localization and molecular characterization of cell membrane receptors for hormones and neurotransmitters. However, such probes are not available for the study of the alpha 2-adrenergic receptor. This report describes the synthesis and characterization of functionalized derivatives of the selective alpha 2-adrenergic antagonists, rauwolscine and yohimbine, which can be radiolabeled to high specific activity with 125I. Following demethylation of rauwolscine or yohimbine, the resultant carboxylic acid derivatives were reacted with 4-aminophenethylamine to yield the respective 4-aminophenethyl carboxamides, 17 alpha-hydroxy-20 alpha-yohimban-16 beta-[N-4-amino-phenethyl]carboxamide (rau-pAPC) and 17 alpha-hydroxy-20 beta-yohimban-16 alpha-[N-4-aminophenethyl]carboxamide. In competitive inhibition studies using rat renal membranes and the radioligand [3H]rauwolscine, rau-pAPC (Ki = 11 +/- 1 nM) exhibited a 14-fold greater affinity than the corresponding yohimbine derivative (Ki = 136 +/- 45 nM). The higher affinity compound, rau-pAPC, was radioiodinated by the chloramine T method, and the product, 125I-rau-pAPC [17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-3 -[125I]iodophenethyl)carboxamide], was purified by reverse phase HPLC to high specific activity (2175 Ci/mmol) and its binding characteristics were investigated in rat kidney membranes. Specific binding of 125I-rau-pAPC was saturable and of high affinity as determined by Scatchard analysis (KD = 1.8 +/- 0.3 nM) or from kinetic studies (KD = k2/k1 = 0.056 +/- 0.013 min-1)/4.3 +/- 0.2 X 10(7) M-1 min-1 = 1.3 +/- 0.3 nM). In competition studies, alpha-adrenergic antagonists and agonists inhibited the binding of 125I-rau-pAPC with a potency order consistent with an interaction at alpha 2-adrenergic receptors (rauwolscine greater than phentolamine greater than prazosin; clonidine greater than (-)-epinephrine greater than (-)-norepinephrine greater than dopamine greater than (+)-epinephrine). In rat liver and human platelet membranes, high affinity binding of 125I-rau-pAPC was also observed (liver, KD = 1.2 +/- 0.4 nM; platelet, KD = 3.2 +/- 1.5 nM). In addition, the density of alpha 2-adrenergic receptors identified from binding studies with 125I-rau-pAPC in kidney, liver, and platelet membranes was similar to that observed in parallel studies with [3H]rauwolscine. These findings indicate that 125I-rau-pAPC is a high affinity probe that selectively identifies alpha 2-adrenergic binding sites. Availability of this radioligand should facilitate the localization and biochemical characterization of this alpha-adrenergic receptor subtype.     

Binding of indolylalkylamines at 5-HT2 serotonin receptors: examination of a hydrophobic binding region

Taking advantage of a proposed hydrophobic region on 5-HT2 receptors previously identified by radioligand-binding studies utilizing various phenylisopropylamine derivatives, we prepared and evaluated several N1 - and/or C7-alkyl-substituted derivatives of alpha-methyltryptamine in order to improve its affinity and selectivity. It was determined that substitution of an n-propyl or amyl group has similar effect on affinity regardless of location (i.e., N1 or C7). The low affinity of several N1-alkylpyrroleethylamines suggests that the benzene portion of the alpha-methyltryptamines is necessary for significant affinity. Whereas tryptamine derivatives generally display little selectivity for the various populations of 5-HT receptors, N1-n-propyl-5-methoxy-alpha-methyltryptamine (3h) binds with significant affinity (Ki = 12 nM) and selectivity at 5-HT2 receptors relative to 5-HT1A (Ki = 7100 nM), 5-HT1B (Ki = 5000 nM), 5-HT1C (Ki = 120 nM), and 5-HT1D (Ki greater than 10,000 nM) receptors. As a consequence, this is the most 5-HT2-selective indolylalkylamine derivative reported to date.     

Cloned human EP1 prostanoid receptor pharmacology characterized using radioligand binding techniques

Prostaglandins such as prostaglandin E2 (PGE2) interact with EP-class prostanoid receptors including EP1, EP2, EP3 and EP4 subtypes. We have conducted a detailed pharmacological characterization of the binding of [3H]-PGE2 to recombinant human EP1 prostanoid receptors expressed in human embryonic kidney (HEK-293) cells using a broad panel of natural and synthetic prostanoids. The receptor displayed high affinity (Kd = 16.0 +/- 0.69 nM; n = 3) for [3H]-PGE2, and was expressed at high levels (Bmax =3.69 +/- 0.30 pmol (mg protein)(-1)) in cell membranes of HEK-293 cells. Specific binding constituted 97.5 +/- 1.4% (n = 12) of the total binding. In competition assays, the rank order of affinities of natural prostanoids for the receptor was PGE2 > PGE1 > PGF2 > PGI2 > PGD2. PGE2 was more effective than PGE1 at displacing bound [3H]-PGE2 (Ki for PGE2 = 14.9 +/- 2.2 nM; Ki for PGE1 = 165 +/- 29 nM). The affinities of enprostil (Ki = 14.5 +/- 3.1 nM) and 17-phenyl-omega-trinor-PGE2 (Ki = 7.3 +/- 2.7 nM) for the receptor were quite similar to that of PGE2, while that of sulprostone (Ki = 137 + 13 nM) more closely resembled PGE1. Some compounds historically classified as specific for DP prostanoid receptors bound with relatively high affinity to the recombinant human EP1 receptor (e.g. ZK118182 (K = 73.4 +/- 8.6 nM) and ZK110841 (K = 166 +/- 20 nM)). All FP (e.g. travoprost acid, fluprostenol), IP (iloprost) and TP (SQ29548) receptor-specific ligands exhibited low affinity (Ki > or = 1 microM).     

Study of species specificity in growth hormone-releasing factor (GRF) interaction with vasoactive intestinal peptide (VIP) receptors using GRF and intestinal VIP receptors from rat and human: evidence that Ac-Tyr1hGRF is a competitive VIP antagonist in the rat

In order to determine species specificity in growth hormone-releasing factor (GRF) interaction with vasoactive intestinal polypeptide (VIP) receptors, we have tested rat (r) GRF (with a His1 such as in VIP), human (h) GRF and position 1 substituted analogs of hGRF (Ala1, Ac-Tyr1, His1, Phe1, and Trp1 in the place of Tyr1) for their ability to inhibit 125I-VIP binding and to stimulate adenylate cyclase activity in human and rat intestinal epithelial membranes. We show that rGRF has a much higher affinity than hGRF for both human and rat VIP receptors. In humans, the Ki values for inhibiting 125I-VIP binding are 0.5 (VIP), 26 (rGRF), and 830 nM (hGRF). In rats the values are 0.6 (VIP), 46 (rGRF), and 1100 nM (hGRF). This is due in part to the presence of His1 in rGRF since the analog His1 hGRF has a higher affinity than hGRF in man and rat, i.e., Ki = 320 nM and 460 nM, respectively. Studies of adenylate cyclase stimulation reveal that rGRF and His1 hGRF are full VIP agonists in man and rat, whereas hGRF and its other analogs behave as partial agonists in both species. One of the hGRF analogs tested (Ac-Tyr1hGRF) is of great interest since it inhibits 125I-VIP binding to rat intestinal membranes with a Ki = 430 nM but has a negligible intrinsic activity in stimulating adenylate cyclase activity (about 6% of the efficacy of VIP). This analog does inhibit the VIP-stimulated adenylate cyclase activity in a dose-dependent manner, complete inhibition of the VIP (0.01-1 nM) effect being obtained with 30 microM analog. The Schild plot of the inhibitory effect further indicates competitive antagonism. In contrast, Ac-Tyr1hGRF is a partial VIP agonist in humans (about 20% of the efficacy of VIP). These results evidence the important role of His1 for peptide interaction with VIP receptors and provide the first example of a competitive VIP antagonist.     

Analogues of the 5-HT1A serotonin antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine with reduced alpha 1-adrenergic affinity.

1-(2-Methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine (NAN-190; 1a) is a putative postsynaptic 5-HT1A serotonin antagonist. This high affinity ligand (Ki = 0.6 nM), although selective for 5-HT1A versus other 5-HT receptors, binds with nearly equal affinity at alpha 1-adrenergic receptors (Ki = 0.8 nm). Structure-affinity relationship studies were conducted in order to achieve an improved selectivity. Replacement of the phthalimide moiety by substituted benzamides led to retention of 5-HT1A affinity but to no improvement in selectivity, whereas replacement by alkyl amides proved beneficial, leading to an improvement in affinity and selectivity. Branching alpha to the amide carbonyl group and increased bulkiness of the alkyl moiety further improved 5-HT1A affinity and selectivity. 4-[4-(1-Adamantanecarboxamido)butyl]-1- (2-methoxyphenyl)piperazine (2j) was found to bind at 5-HT1A sites with high affinity (Ki = 0.4 nM) and with a 160-fold selectivity over alpha 1-adrenergic sites. Preliminary studies show that this agent retains antagonist activity as determined in a 5-HT1A-coupled adenylyl cyclase assay. Further functional studies are warranted to fully characterize this agent.