DAU 5884 hydrochloride

Pharmacological analyses of endo-6-methoxy-8-methyl-8-azabicyclo[3.2.1]oct-3-yl-2,3-dihydro-2-oxo-1 H- benzimidazole-1-carboxylate hydrochloride (DAU 6285) at the 5-hydroxytryptamine4 receptor in the tunica muscularis mucosae of rat esophagus and ileum of guinea pig: role of endogenous 5-hydroxytryptamine.[Pubmed:8437113]

J Pharmacol Exp Ther. 1993 Feb;264(2):654-61.

Functional estimates of affinity for endo-6-methoxy-8-methyl-8- azabicyclo[3.2.1]oct-3-yl-2,3-dihydro-2-oxo-1H-benzimidazole-1-carboxyla te hydrochloride (DAU 6285) were made at the 5-hydroxytryptamine4 (5-HT4) receptor in isolated preparations of rat esophageal tunica muscularis mucosae (TMM) and guinea pig ileum. In the TMM, relaxation of carbachol-induced contracture by 5-HT4 receptor agonism of longitudinal muscle was recorded. Estimated pA2 values for DAU 6285 of 6.9 to 7.2 were tissue, time (1-3 hr equilibration) and agonist-independent. However, DAU 6285 increased the maximal response to 5-HT and 5-methoxytryptamine in the TMM and augmented the contractile tone to carbachol. These effects were not observed in guinea pig ileum, suggesting a tissue-dependent mechanism. [3a-Tropanyl]-1H-indole-3-carboxylic acid ester (tropisetron) and 2-methoxy-4-amino-5-chloro-benzoic acid 2-(diethylamino)ethyl ester (SDZ 205-557), two other 5-HT4 receptor antagonists, mimicked the effects of DAU 6285. Mechanistic experiments suggest agonism by endogenous 5-HT, within the isolated TMM, to explain the effects of 5-HT4 receptor antagonists. Pretreatment of rats with parachlorophenylalanine to deplete endogenous 5-HT, prevented the effect of DAU 6285 on the maximal response to 5-HT and carbachol-induced tone. In conclusion, DAU 6285 acts as a silent, competitive antagonist at 5-HT4 receptors in rat TMM and guinea pig ileum. However, in the TMM, endogenously released 5-HT confounds interpretation. The TMM, as a quantitative assay system for 5-HT4 receptor agonists and antagonists may be improved by pretreating rats with parachlorophenylalanine.

Muscarinic M(3) receptor-dependent regulation of airway smooth muscle contractile phenotype.[Pubmed:14993104]

Br J Pharmacol. 2004 Mar;141(6):943-50.

1. Airway smooth muscle (ASM) cells are known to switch from a contractile to a proliferative and synthetic phenotype in culture in response to serum and growth factors. Phenotype switching in response to contractile agonists, however, is poorly characterised, despite the possible relationship between ASM phenotype and airway remodelling in asthma. 2. To investigate the effects of muscarinic receptor stimulation on ASM phenotype, we used organ-cultured bovine tracheal smooth muscle (BTSM) strips, in which contractile responsiveness, contractile protein expression and proliferation were measured after pretreatment with methacholine. 3. Long-term methacholine pretreatment (8 days) decreased maximal contraction and sensitivity to methacholine as well as to histamine and KCl. This decrease was dose-dependent (pEC(50)=5.2+/-0.1). Pretreatment with the highest concentration of methacholine applied (100 microm) could suppress maximal histamine-induced contraction to 8+/-1% of control. In addition, contractile protein expression (myosin, actin) was downregulated two-fold. No concomitant increase in proliferative capacity was observed. 4. The M(3)/M(2) muscarinic receptor antagonist DAU 5884 (0.1 microm) completely inhibited the observed decrease in contractility. In contrast, the M(2)/M(3) muscarinic receptor antagonist gallamine (10 microm) was ineffective, demonstrating that M(2) receptors were not involved. 5. Pretreatment (8 days) with 60 mm KCl could mimick the strong decreases in contractility. This was completely prevented by pretreatment with verapamil (1 microm). 6. Regulation of contractility was not affected by protein kinase C inhibition, whereas inhibitors of phosphatidyl inositol 3-kinase and p42/p44 mitogen activated protein kinase were partially effective. 7. These results show that long-term methacholine pretreatment (8 days) induces an M(3) receptor-dependent decrease in BTSM contractility without increased proliferative capacity.

Muscarinic M3-receptors mediate cholinergic synergism of mitogenesis in airway smooth muscle.[Pubmed:12540494]

Am J Respir Cell Mol Biol. 2003 Feb;28(2):257-62.

Muscarinic receptor agonists have been considered to act synergistically in combination with growth facors on airway smooth muscle growth. Characterization of the proliferative responses and of the receptor subtype(s) involved has not yet been studied. Therefore, we investigated mitogenesis induced by stimulation of muscarinic receptors, alone and in combination with stimulation by platelet-derived growth factor (PDGF). For this purpose, [(3)H]thymidine-incorporation was measured at different culture stages in bovine tracheal smooth muscle cells. Functional muscarinic M(3)-receptors, as measured by formation of inositol phosphates, were present in unpassaged cells, but were lacking in passage 2 cells. Methacholine (10 microM) by itself was not able to induce a proliferative response in both cell culture stages. However, methacholine interacted synergistically with PDGF in a dose-dependent fashion (0.1-10 microM), but only in cells having functional muscarinic M(3)-receptors. This synergism could be suppressed significantly by the selective M(3)-receptor antagonists DAU 5884 (0.1 microM) and 4-DAMP (10 nM), but not at all by the M(2)-subtype selective antagonist gallamine (10 microM). These results show that methacholine potentiates mitogenesis induced by PDGF solely through stimulation of muscarinic M(3)-receptors in bovine tracheal smooth muscle cells.

Characterization of muscarinic receptors in guinea-pig uterus.[Pubmed:8112383]

Eur J Pharmacol. 1993 Dec 7;250(2):223-30.

To characterize the muscarinic receptor present in guinea-pig uterus smooth muscle the affinities of a series of 27 muscarinic receptor antagonists for M1 (rat cortex), M2 (rat heart), M3 (rat submandibular gland), m4 (transfected in CHO cells) and muscarinic binding sites in guinea-pig uterus smooth muscle were determined in radioligand binding studies. In addition, functional experiments were performed to assess pKB values of the antagonist for muscarinic receptors in guinea-pig atrium and uterus. The results obtained are consistent with the presence of M2 receptors in the uterus through which the functional contractile response is mediated. Correlation coefficients of 0.98, 0.91 and 0.91 were calculated for the following linear regressions: pKi uterus vs. pKi M2, pKB uterus vs. pKi M2 and pKB uterus vs. pKB atrium. This study also revealed that the compounds dicyclomine, DAU 5884, DAU 6202 as well as AQ-RA 721 could distinguish m4 from M2 sites and are therefore important tools to characterize muscarinic receptor subtypes. In addition, DAU 5884 and DAU 6202 have been identified as highly potent M1 selective antagonists.

Cholinergic contraction of the guinea pig lung strip is mediated by muscarinic M2-like receptors.[Pubmed:8112384]

Eur J Pharmacol. 1993 Dec 7;250(2):267-79.

The muscarinic receptor subtype mediating contraction of the guinea pig lung strip preparation was investigated and compared with that in guinea pig tracheal and human peripheral airway (small bronchi) smooth muscle preparations, using a number of subtype selective muscarinic receptor antagonists. It was found that guinea pig lung strip contraction was not mediated by a homogeneous class of muscarinic M3 receptors, in contrast to guinea pig tracheal and human peripheral airway smooth muscle. The affinities of the M1- and M3/M2-selective muscarinic receptor antagonists on the guinea pig lung strip were between 0.35 and 1.94 log units lower than in the M3 receptor tissues (respective pA2 values on guinea pig lung strip and trachea: pirenzepine 6.36/6.71, AF-DX 474 6.39/7.11, AQ-RA 721 6.93/7.96, DAU 5884 6.78/8.72, UH-AH 371 7.04/8.20), whereas the affinities of the M2/M3-selective antagonists were between 0.63 and 1.97 log units higher (AF-DX 116 6.63/6.00, AQ-RA 741 7.48/6.63, gallamine 5.44/3.47, methoctramine 7.30/5.38). As a result, a good correlation was obtained when pA2 values from guinea pig lung strip were compared to pKi values towards bovine cardiac muscarinic M2 receptors, though it was noticed that pirenzepine and the M3/M2-selective antagonists showed a closer relationship than the M2-selective compounds. These results suggest that cholinergic contraction of the guinea pig lung strip is mediated by muscarinic M2-like receptors, possibly representing a novel subtype or a mixture of M2 (cardiac) and M3 (or M4) subtypes. It remains to be established, however, on what structure in the lung these contractile M2-like receptors are located and also by which transduction mechanism they produce contraction.