Ettore Giraldo

Binding profile of a novel cardioselective muscarine receptor antagonist, AF-DX 116, to membranes of peripheral tissues and brain in the rat

The heterogeneity of muscarine receptors was examined in two brain regions (cerebral cortex and cerebellum) and in some parasympathetically innervated peripheral tissues (heart, salivary gland and intraorbital lacrimal gland), by in vitro binding techniques. As a tool, we used a new antimuscarinic compound, AF-DX 116 (see text for structural formula and chemical name). In competition experiments against 3H-N-methylscopolamine (3H-NMS) or 3H-pirenzepine (3H-PZ), AF-DX 116 was found to bind with high affinity to muscarine receptors in the heart and cerebellum (KDs approximately equal to 115 nM), with intermediate affinity to M1 receptors in neuronal tissue (KD = 760 nM) and with low affinity to receptors in exocrine glands (KDs approximately equal to 3200 nM). Its receptor interaction was found to be of the simple, competitive type. Thus, AF-DX 116 shows a novel cardioselective profile. On the basis of the results which demonstrate that the muscarine receptors in the heart and exocrine glands are clearly distinct, it is proposed that these receptors may be subclassified as M2 cardiac type and M2 glandular type muscarine receptors.

Distribution of muscarinic receptor subtypes in rat brain as determined in binding studies with AF-DX 116 and pirenzepine

In vitro competition binding experiments with the selective muscarinic antagonists AF-DX 116 and pirenzepine (PZ) vs 3H-N-methylscopolamine as radioligand revealed a characteristic distribution of muscarinic receptor subtypes in different regions of rat brain. Based on non linear least squares analysis, the binding data were compatible with the presence of three different subtypes: the M1 receptor (high affinity for PZ), the cardiac M2 receptor (high affinity for AF-DX 116) and the glandular M2 receptor (low affinity for PZ and AF-DX 116). The highest proportion of M1 receptors was found in the hippocampus, whilst the cerebellum and the hypothalamus were the regions with the largest fraction of the cardiac M2 and glandular M2 receptors, respectively. In certain brain areas, depending on the relative proportions of the subtypes, flat binding curves were seen for AF-DX 116 and PZ. Based on these data, an approximate distribution pattern of the subtypes in the various brain regions is presented.

Muscarinic receptor heterogeneity in guinea pig intestinal smooth muscle: binding studies with AF-DX 116

Muscarinic receptor subtypes in longitudinal and circular smooth muscles of the guinea pig ileum were characterized with the use of the cardioselective antagonist AF-DX 116 in binding competition experiments against 0.3 nM [3H] N-methylscopolamine [( 3H]NMS). This compound recognized a heterogeneous receptor population in both smooth muscles, revealing the existence of different percentages of the cardiac (KD = 92-110 nM) and the glandular (KD = 1150-2541 nM) muscarinic receptor subtypes. These results, together with the low potency displayed by AF-DX 116 to inhibit the agonist-stimulated smooth muscle contraction and salivary secretion allow the suggestion that the glandular muscarinic receptor subtype, showing a low affinity for AF-DX 116, is involved in smooth muscle contraction.

Characterization of the muscarinic receptor subtypes in the rat urinary bladder

We investigated the nature of the muscarinic receptors present in the rat urinary bladder by performing binding studies with various selective (pirenzepine, AF-DX 116, hexahydrosiladifenidol, benzhexol, 4-diphenyl-acetoxy-N-methyl piperidine methiodide, dicyclomine, secoverine) and classical (N-methylscopolamine, atropine) antagonists. Competition experiments were carried out against [3H]N-methyl scopolamine at 30 degrees C in Na+/Mg2+ HEPES buffer; non-specific binding was determined in the presence of 1 microM 3-quinuclidinyl benzilate. Of all the antagonists examined, only AF-DX 116 exhibited a heterogeneous binding profile (nH less than 1). Computer-assisted analysis showed that the data fitted best to a two-binding site model, revealing the existence of high and low affinity receptors. The affinity values of AF-DX 116, determined in binding experiments carried out in heart and gland homogenates, allowed us to classify the rat urinary bladder receptors into cardiac and glandular subtypes. We suggest that the glandular receptor subtype is involved in smooth muscle contraction, since AF-DX 116 was equally potent in inhibiting smooth muscle contraction and the secretion of saliva.

BIMT 17, a 5-HT2A receptor antagonist and 5-HT1A receptor full agonist in rat cerebral cortex

In the search for antidepressant agents with a rapid onset of action, we have found that compound BIMT 17 (1-[2-[4-(3-trifluoromethylphenyl)piperazin1-yl]ethyl]benzimidazol-[1H]-2-one) shows a good affinity for cerebral cortical 5-HT1A (pKi = 7.72) and 5-HT2A (pKi = 6.90) receptors, with no appreciable affinity for the other 5-HT receptor subtypes, including 5-HT2C. BIMT 17 reduced forskolin-stimulated cAMP accumulation in the cerebral cortex (pEC50 = 6.09) and in the hippocampus (pEC50 = 6.50), and antagonized 5-HT-induced phosphatidylinositol turnover (pKi = 6.96) in the cerebral cortex. The effect on cAMP accumulation was blocked by the 5-HT1A receptor antagonist tertatolol. Buspirone, 8-OH-DPAT and S 14671 {1-[2-(2-thenoylamino)ethyl]-4[1-(7-methoxynaphtyl)]piperazine, claimed to be 5-HT1A receptor agonists, did not reduce forskolin-stimulated cAMP formation in the cerebral cortex.On the basis of these data, it was concluded that BIMT 17 was the only compound that behaved as a full agonist with respect to the CAMP response in the cortex, while exerting concurrent agonism at 5-HT1A receptors and antagonism at 5-HT2A receptors. These characteristics might explain the peculiar behaviour of BIMT 17 in mimicking the inhibitory action of 5-HT on the basal firing rate of the cortical neurons (see accompanying paper).

Pharmacological characterization of the 5-hydroxytryptamine receptor coupled to adenylyl cyclase stimulation in human brain

Recently, a 5-hydroxytryptamine (5-HT) receptor has been described, whose pharmacology was distinct from that of the already known serotonergic receptors, so that it has been called 5-HT4. Because the lack of a high affinity radioligand, the identification of this receptor depends entirely on functional pharmacological analysis. Its stimulation leads to an increase in cyclic AMP accumulation in mouse embryo colliculi neurons, in guinea pig hippocampus and in human heart. We studied the effect of two indoleamines, 5-HT and 5-methoxytryptamine (5-MeO-T), and a benzimidazolone derivative, BIMU 8, in stimulating basal adenylyl cyclase activity in human frontal cortex, and characterized the receptor subtype involved. In membranes prepared from this tissue, 5-HT, 5-MeO-T and BIMU 8 dose-dependently stimulated (13-25%) the basal enzyme activity (220 pmoles cyclic AMP/min/mg protein). 5-MeO-T behaved as a full agonist, BIMU 8 elicited about 60% of the maximal 5-HT effect. The selective 5-HT1A agonist 8-OH-DPAT, was devoid of any stimulating activity. ICS 205-930, a low affinity 5-HT4 receptor antagonist, completely reversed the effect of all three agonists at high concentrations. Therefore, the present data are consistent with the 5-HT-mediated stimulation of adenylyl cyclase in human frontal cortex resulting by the activation of a 5-HT4 receptor subtype.

DAU 6285: A novel antagonist at the putative 5-HT4 receptor

The antagonistic properties of DAU 6285, an azabicycloalkyl benzimidazolone derivative, at putative 5-hydroxytryptamine4 (5-HT4) receptors were investigated in in vitro preparations of guinea-pig ileum and human atrium, in comparison to ICS 205-930. DAU 6285 behaved as a competitive antagonist in all the preparations examined. Its affinity (pA2) ranged between 6.50 and 7.12 in the test models considered. The affinity of ICS 205-930 was 2-3 fold lower. At variance with ICS 205-930, DAU 6285 displayed a weak affinity for 5-HT3 receptors (pKi = 6.1, rat cortex; pA2 less than 5, guinea-pig ileum). In the guinea-pig ileum, DAU 6285 (10 microM) did not exert antimuscarinic, antihistaminic, antinicotinic or myolytic activity. Moreover, it did not bind to other 5-HT receptor subtypes, or to adrenergic, dopaminergic, benzodiazepine, nicotine, GABA receptors. DAU 6285 may represent a suitable tool for studies in the field of 5-HT4 receptors.

Characterization of muscarinic receptors in salivary and lacrimal glands of the rat.

The binding characteristics of muscarinic receptors in rat salivary and lacrimal glands were studied by means of radioligand binding techniques. In competition experiments against [3H]N-methylscopolamine, classical muscarinic antagonists ipratropium bromide, N-methylscopolamine and N-methylatropine exhibited very similar KD values in all the glands and their binding behavior was well described by a one binding site model (nH congruent to 1). The novel cardioselective antimuscarinic compound, AF-DX 116, displayed an equally low affinity in all the tissues examined. Pirenzepine and dicyclomine, two other selective muscarinic antagonists, showed a similar behaviour in all but the sublingual gland, where their binding profile indicated the presence of a heterogeneous receptor population (nH = 0.74 and 0.84, respectively). Histological studies of the sublingual-submandibular glandular complex demonstrated the presence of ganglionic structures mainly located in the hilum of the sublingual-submandibular glandular complex connected with the sublingual gland. Binding studies carried out with pirenzepine on the hilum and on a synaptosomal preparation from this region again revealed the presence of two populations of muscarinic receptors with KD values of 22-25 and 270-463 nM. These results are best explained by the presence of M1 and M2 receptors located on neuronal and glandular structures.

The role of electronic and conformational properties in the activity of 5-HT3 receptor antagonists

Abstract The conformational and electronic properties of 11 5-HT 3 receptor antagonists, were investigated to rationalize their biological activity. All compounds fulfill the previously reported geometrical requirements for the pharmacophore, although they show a wide spectrum of activities. Quantitative structure-activity relationships (QSAR) models were searched using the cross-validated partial least squares technique. The results of the QSAR analysis show that a unique model can rationalize both the in vitro (affinity, K d ) and the in vivo (50% inhibitory dose, ID 50 ) activities. This model includes the value of the molecular electrostatic potential minimum above the aromatic fragment, used as an index of the π-electron density, as well as the relative population of the anti-periplanar (app, the “active”) conformation. Two other models were also obtained that include electrophilic superdelocalizability indexes. Although these models give a more satisfactory value of the cross-validated correlation coefficient ( R 2 cv ), they represent two different models for K d and ID 50 which is in contrast with the experimental evidence that a correlation exists between these two activities. It is concluded that the use of electronic and conformational properties in addition to the geometrical characteristics may better describe and predict the activity of 5-HT 3 receptor antagonists, than the use of geometrical characteristics alone.

Guanylpirenzepine distinguishes between neuronal ml and m4 muscarinic receptor subtypes

Guanylpirenzepine, a polar, non-quaternary analog of pirenzepine, exhibited a novel binding behavior in rat brain regions: in competition binding experiments against [3H]pirenzepine labeling the M1 receptor in membranes from cerebral cortex, hippocampus and striatum, the compound, differently from pirenzepine, displayed heterogeneous binding curves. Computer assisted analysis of these curves, evidenced the existence of two populations of binding sites: a large proportion (84-89%) of high affinity receptors (KH = 64-92 nM) and a remainder with very low affinity (KL = 19-28 microM). Like pirenzepine, guanylpirenzepine showed low affinity for the glandular M3 and the cardiac M2 receptors when [3H]N-methylscopolamine was used to label the receptors in membranes from these two tissues; affinity values for guanylpirenzepine were 1336 and 5790 nM respectively, vs 323 and 683 nM for pirenzepine. We conclude that guanylpirenzepine is able to discriminate between m1 and m4 receptor subtypes and may represent a new tool for deeper studies on muscarinic receptors classification.