Characterization of the muscarinic receptor subtype in isolated gastric fundic cells of the rabbit.

The characteristics of the muscarinic receptor in isolated gastric fundic cells from rabbit were determined by radioligand binding techniques and functional tests. The dissociation constants (KDS) of selective (hexahydrosiladifenidol and pirenzepine) and non-selective (N-methylscopolamine and atropine) muscarinic receptor antagonists obtained in competition experiments vs [3H]-N-methylscopolamine were compared with the pA2 values of the drugs as inhibitors of carbachol-stimulated [14C]-aminopyrine accumulation (an index of acid secretion) in the gastric fundic cells. Good correlations were found between the ability of the drugs to inhibit acid secretion and their affinity for muscarinic receptors in the gastric fundic cells. The rank order of potency in both tests was N-methylscopolamine greater than atropine greater than hexahydrosiladifenidol greater than pirenzepine. The character of the muscarinic receptor subtype present on gastric fundic cells was established by comparing the affinity values of the compounds for this receptor with those for the receptors in other rabbit tissues. It was found that only pirenzepine and hexahydrosiladifenidol displayed tissue selectivity in their binding profiles. The KDS for pirenzepine were 13nM for the M1 receptor of the cerebral cortex and about 500 nM for the M2 receptors of the submandibular and gastric glands and heart. Differently from pirenzepine, hexahydrosiladifenidol showed about 10-fold discrimination between the M2 subtype of the gland (KD = 31 nM) and the M2 subtype of the heart (KD = 330 nM).

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.

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 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.

Azabicycloalkyl benzimidazolone derivatives as a novel class of potent agonists at the 5-HT4 receptor positively coupled to adenylate cyclase in brain.

Recent experimental evidence indicates that central 5-HT4 receptors which are positively coupled to adenylate cyclase, are stimulated by a family of 2-methoxy-4-amino-5-chloro substituted benzamide derivatives. These compounds are also potent stimulants of the gastro-intestinal motility. In this study the ability of three azabicycloalkyl benzimidazolone derivatives, BIMU 1, BIMU 8, and DAU 6215 (structural formulas are given in the text), to stimulate cAMP formation in colliculi neurons in primary culture have been tested. Two of the compounds, BIMU 1 and BIMU 8, which show prokinetic activity in various animal models, were also good agonists at the 5-HT4 receptors, whereas DAU 6215, a drug devoid of prokinetic activity, was only a weak, partial agonist at 5-HT4 receptors. The rank order of their potencies as compared with those of 5-HT and cisapride was as follows: BIMU 8 = cisapride greater than 5-HT greater than BIMU 1 greater than DAU 6215. The efficacies of BIMU 8 and cisapride were comparable (133 +/- 9% and 124 +/- 8% of the maximal 5-HT efficacy, respectively), whereas BIMU 1 and DAU 6215 elicited, respectively, only 72 +/- 11% and 16 +/- 4% of the maximal 5-HT effect. The activities of the azabicycloalkyl benzimidazolone derivatives and 5-HT on cAMP formation were not additive and ICS 205-930 antagonized the stimulatory effect of these compounds with low potency (pKi = 6.1-6.4), further strengthening the notion of interaction with 5-HT4 receptors. In addition, cross desensitization between the effects of 5-HT and the azabicycloalkyl benzimidazolones on adenylate cyclase was noted, another argument in favor of an interaction of these drugs on 5-HT4 receptors.

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)piperazin-1- 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 behavior of BIMT 17 in mimicking the inhibitory action of 5-HT on the basal firing rate of the cortical neurons (see accompanying paper).

Characterization of a novel 5-HT4 receptor antagonist of the azabicycloalkyl benzimidazolone class: DAU 6285.

Three chemical classes of serotonin 5-HT4 receptor agonists have been identified so far: 5-substituted indoles (e.g. 5-HT), benzamides (e.g. renzapride) and benzimidazolones (e.g. BIMU 8). In a search for 5-HT4 receptor antagonists, we have discovered that the benzimidazolone derivative DAU 6285 (for structure see text), is 3-5 times more potent than tropisetron in blocking 5-HT, renzapride and BIMU 8 induced stimulation of adenylate cyclase activity in mouse embryo colliculi neurons. Schild plot analysis yielded Ki values of 220, 181 and 255 nmol/l, respectively. In addition, DAU 6285 showed poor activity as a 5-HT3 receptor ligand with respect to tropisetron, as demonstrated by in vitro binding studies (Ki, 322 vs 2.8 nmol/l) and by its antagonistic activity in the Bezold-Jarisch reflex test (ID50, 231 vs 0.5 micrograms/kg, i.v.). No significant binding (Ki greater than 10 mumol/l) of DAU 6285 to serotonergic 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D, and 5-HT2 receptors as well as to adrenergic alpha 1, alpha 2, dopaminergic D1, D2 or muscarinic M1-M3 receptor subtypes was found. The data indicate that DAU 6285 has a somewhat higher affinity than tropisetron for 5-HT4 receptors, a property confirmed in functional tests, and much lower affinity than tropisetron for 5-HT3 receptors. The compound represents a new interesting tool for investigating the pharmacological and physiological properties of 5-HT4 receptors.

Binding characteristics of mu and kappa agonists in rat brain subcellular fractions.

Scatchard analysis of 3H-dihydromorphine (3H-DHM) binding to rat brain subcellular fractions gave a curvilinear plot in the microsomal (P3) and a linear plot in the mitochondrial (P2) fraction, respectively. In contrast, plots of 3H-ethylketocyclazocine (3H-EK) binding were linear in the P3 and curvilinear in the P2 fraction. No differences were seen in the potency of morphine and ketazocine in displacing either 3H-DHM or 3H-EK in the P2 fraction. In the P3 fraction, however, morphine was more potent than ketazocine in displacing 3H-DHM, but the reverse was true when 3H-EK was used as the ligand. Isolation of the P3 fraction may facilitate the demonstration of a kappa receptor in the rat brain.

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 (KD's 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 (KD's 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.

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.

Identification of a novel polymorphism in the fibronectin type II domain of the SEL1L gene and possible relation to the persistent hyperinsulinemic hypoglycemia of infancy.

SEL1L, a human gene located on chromosome 14q24.3-q31, is highly expressed in adult pancreas. It is proximal to D14S67 (IDDM11) a proposed type I diabetes susceptibility locus. Considering the organ specific expression of SEL1L, a fundamental role of SEL1L in pancreatic growth can be hypothesized. While screening for mutations in young diabetic patients, in children affected by persistent hyperinsulinemic hypoglycemia of infancy (PHHI), in patients with non-functional endocrine tumours and in over 100 control subjects, we identified a novel polymorphism (D162G) residing on the fourth exon of the gene. This exon encodes for the fibronectin type II domain and the nucleotide change involves a highly conserved amino acid. The D162G polymorphism induces a major change in the amino acid composition producing a possible disruptive role in collagen binding.

New CCK antagonists derived from the amino acid residues of the CCK-8 fragment.

The synthesis of lipophylic derivatives of the amino acid residues of the CCK-8 fragment is described. According to "in vitro" binding studies and functional test, nearly all the compounds behaves as CCK-antagonists; moreover some compounds are able to interact differentially with CCK-A and CCK-B receptor subtype. In particular, compounds 2c, 2g, and 2h possess a high affinity for the CCK-A receptor subtype coupled with a low affinity for the CCK-B subtype. This results in an interesting selectivity profile. However, the same compounds are not able to antagonize the effects exerted by CCK-itself, when tested in "in vivo" assays.

Medicinal chemistry of muscarinic agonists for the treatment of dementia disorders.

Alzheimer disease (AD) is a neurodegenerative disorder lacking an effective therapy. The etiology is controversial and among different drug strategies, the cholinergic approach has gained great interest owing to biochemical and pharmacological evidence of the crucial role of acetylcholine in cognitive functions. Several attempts exploiting the boosting of the cholinergic system are currently under way. Inhibitors of the acetylcholinesterase enzyme sustain the availability of the natural transmitter by limiting its removal from the synapse. In a different approach, exogenous agonists may substitute acetylcholine itself. In this way the issue of the extensive cholinergic cell loss occurring in AD and leading to a reduction of cholinergic functions, could be advantageously bypassed. Moreover the discovery of different muscarinic receptor subtypes, most notably the M1 subtype as that involved in the postsynaptic transmission, has offered new opportunities to face the problem in a very specific way. In this line of research, we have now identified BIMC 182 as a new functionally selective M1 agonist. Whereas its affinity for the different receptor subtypes is almost similar (radioreceptor binding), its functional selectivity is pointed out by specific "in vitro" models. BIMC 182 behaves as a full agonist at M1 (rat superior cervical ganglion, pD2 4.8) and as a partial agonist at M2 and M3 sites (g.p. heart pD2 = 5.4 and g.p. ileum pD2 = 4.5). The agonist profile is further confirmed in hm1 transfected CHO cells where the compound stimulates PI turnover. BIMC 182 penetrates well the brain as shown by the increase in the energy of the low frequency band (theta waves) in the cortical EEG of rabbits (3 mg/kg i.v.).(ABSTRACT TRUNCATED AT 250 WORDS)

Ctla4 and multiple sclerosis in the Italian population.

CTLA4 protein is a receptor molecule that plays a critical role as a negative regulator of the immune response. Therefore, genetic variations in CTLA4 may confer susceptibility to autoimmune diseases such as multiple sclerosis (MS). In order to investigate the association of two CTLA4 polymorphisms (+49 A/G and -318 C/T) with multiple sclerosis, sporadic MS patients and healthy controls from Italy were genotyped through direct DNA sequencing. Considering single-loci variations, no differences in the allelic and genotypic frequencies between patients and controls were found. However, considering a putative interaction at the two loci, the T/G combination was more frequently observed in patients than in controls. This result suggests that this allelic combination of the CTLA4 polymorphisms may be involved in the susceptibility to MS in the Italian population.

Selective labelling of M1-receptors in autonomic ganglia with 3H-pirenzepine.

Using the classical muscarinic antagonist 3H-N-methyl-scopolamine as radioligand and unlabelled pirenzepine (PZ) as displacing agent, a heterogeneous muscarinic receptor population consisting of about 70% M1-receptors and 30% M2-receptor, can be demonstrated in crude membranes of calf sympathetic ganglia. In the same preparation only low and variable specific binding is detectable when 3H-PZ is used as marker ligand. The situation clearly improves when synaptosomes are prepared from the ganglia. Then binding of 3H-PZ can be measured with a favourable specific to non specific binding ratio and with features compatible with the selective labelling of ganglionic M1-receptor sites. It is concluded that the 3H-PZ binding assay in synaptosomes of autonomic ganglia is a novel method for the characterization of the peripheral M1-receptor.