Investigation of the relationship between cell-surface calcium-ion gating and phosphatidylinositol turnover by comparison of the effects of elevated extracellular potassium ion concentration on ileium smooth muscle and pancreas. (1/35)

Incubation of fragments of guinea-pig ileum smooth muscle in the presence of an elevated extracellular K+ concentration, which causes an increase in cell-surface Ca2+ permeability and thus leads to contraction, caused a marked increase in phosphatidylinositol turnover, as assessed by incorporation of 32Pi. This response was not diminished by atropine or propylbenzilycholine mustard, two muscarinic cholinergic antagonists, and was therefore not caused by the release of endogenous acetylcholine within the tissue. In contrast, exposure of guinea-pig pancreas fragments to high extracellular [K+], which does not increase cell-surface Ca2+ permeability or evoke secretion, did not cause an increase in phosphatidylinositol turnover, even though such an increase was triggered by carbamoylcholine, which is a secretagogue. These observations are consistent with a suggested function for phosphatidylinositol breakdown in the mechanisms of cell-surface Ca2+ gates.  (+info)

Evidence for a tandem two-site model of ligand binding to muscarinic acetylcholine receptors. (2/35)

After short preincubations with N-[(3)H]methylscopolamine ([(3)H]NMS) or R(-)-[(3)H]quinuclidinyl benzilate ([(3)H]QNB), radioligand dissociation from muscarinic M(1) receptors in Chinese hamster ovary cell membranes was fast, monoexponential, and independent of the concentration of unlabeled NMS or QNB added to reveal dissociation. After long preincubations, the dissociation was slow, not monoexponential, and inversely related to the concentration of the unlabeled ligand. Apparently, the unlabeled ligand becomes able to associate with the receptor simultaneously with the already bound radioligand if the preincubation lasts for a long period, and to hinder radioligand dissociation. When the membranes were preincubated with [(3)H]NMS and then exposed to benzilylcholine mustard (covalently binding specific ligand), [(3)H]NMS dissociation was blocked in wild-type receptors, but not in mutated (D99N) M(1) receptors. Covalently binding [(3)H]propylbenzilylcholine mustard detected substantially more binding sites than [(3)H]NMS. The observations support a model in which the receptor binding domain has two tandemly arranged subsites for classical ligands, a peripheral one and a central one. Ligands bind to the peripheral subsite first (binding with lower affinity) and translocate to the central subsite (binding with higher affinity). The peripheral subsite of M(1) receptors may include Asp-99. Experimental data on [(3)H]NMS and [(3)H]QNB association and dissociation perfectly agree with the predictions of the tandem two-site model.  (+info)

Characterization of subtype of propylbenzilylcholine mustard (PrBCM)-sensitive and -resistant muscarinic cholinoceptors in guinea pig ileal muscle. (3/35)

The subtype of propylbenzilylcholine mustard (PrBCM)-sensitive and -resistant muscarinic cholinoceptors in guinea pig ileal muscle was examined using four selective muscarinic antagonists, pirenzepine, AF-DX 116, himbacine and 4-DAMP. The pA2 values of the four antagonists against pilocarpine were not different from their values against carbachol after the treatment with PrBCM and was identified with the values for the m3 subtype. These results suggest that the subtype of PrBCM-sensitive and -resistant muscarinic cholinoceptors in guinea pig ileal muscle is the m3 subtype only and not other subtypes.  (+info)

Guanosine 5'-triphosphate converts some populations of propylbenzilylcholine mustard-sensitive muscarinic cholinoceptor sites to sites resistant to the drug in intestinal smooth muscle. (4/35)

From functional studies with propylbenzilylcholine mustard (PrBCM), we reported that there coexist PrBCM-sensitive and PrBCM-resistant muscarinic cholinoceptor mechanisms in guinea pig taenia caecum. We investigated the interrelationship between these two cholinoceptor mechanisms using an in vitro receptor binding assay with [3H]quinuclidinyl benzilate (QNB) and [3H]PrBCM. Pretreatment of the muscle strips with 300 nM PrBCM (in vivo alkylation) for 10-50 min resulted in progressive decreases of the number of the maximum [3H]QNB binding sites. However, a prolongation of the period of in vivo alkylation up to 90 min was accompanied with no further loss in the binding sites. Under these conditions, there is no significant change in the affinity of [3H]QNB for the binding sites. The concentration of carbachol required to displace 50% of the bound [3H]QNB was larger in membranes obtained from the tissues that had been alkylated in vivo with PrBCM for 50 min than that from control strips, but was not altered when the pretreatment with the drug was carried out after homogenization (in vitro alkylation). When GTP was added during in vitro alkylation, the affinity of carbachol was lower than that in control membranes, as observed when in vivo alkylation was carried out. In the presence of guanine nucleotide, PrBCM thus appears to recognize two distinct populations or states of muscarinic receptors.  (+info)

A difference in receptor mechanisms for muscarinic full and partial agonists. (5/35)

Concentration-response curves of 4 muscarinic full agonists were progressively inhibited by 10 to 50-min treatments of the longitudinal muscle of guinea pig ileum with propylbenzilylcholine mustard (PrBCM, 3 x 10(-6) M). A 90-min treatment with PrBCM had no further significant inhibitory effect on their curves. The 50-min treatment with PrBCM (3 x 10(-6) M) completely inhibited the concentration-response curves of 6 partial agonists. The limiting effect of PrBCM observed on the concentration-response curves of the full agonists was not found on the curves of the partial agonists. These results suggest that there are two subtypes of M3-cholinoceptors, PrBCM-sensitive receptors and PrBCM-resistant ones. Pilocarpine, a partial agonist, shifted the concentration-response curve of carbachol, a full agonist, in a parallel fashion in the strips treated with PrBCM (3 x 10(-6) M) for 50 min, suggesting that an interaction of pilocarpine with PrBCM-resistant cholinoceptors does not induce contraction. The full agonists contract the longitudinal muscle through the interaction of two cholinoceptors, PrBCM-sensitive and -resistant ones, while the partial agonists produce the contraction through the activation of PrBCM-sensitive ones.  (+info)

Propylbenzilycholine mustard (PrBCM)-sensitive cholinoceptors and contractile response to partial agonist in guinea pig ileal muscle. (6/35)

Pilocarpine, a partial agonist, activates propylbenzilylcholine mustard (PrBCM)-sensitive cholinoceptors in the guinea pig ileal longitudinal muscle, while carbachol, a full agonist, predominantly activates PrBCM-resistant ones. Carbachol behaves as a partial agonist in the preparation treated with phenoxybenzamine and mainly activates PrBCM-sensitive cholinoceptors, as phenoxybenzamine preferably blocks PrBCM-resistant ones. The receptor occupancy-response curve for carbachol became a rectangular hyperbola, while pilocarpine showed a linear relation. After occlusion of cholinoceptors with phenoxybenzamine, carbachol showed a linear receptor occupancy-response relation, suggesting that its contraction mechanisms after occlusion of cholinoceptors resemble those for pilocarpine. Both the agonists induced an increase in cytosolic Ca2+ concentration [( Ca2+]i) and tension development in a concentration-dependent manner under the conditions used herein. The slopes of the regression lines between [Ca2+]i and tension development for pilocarpine in the untreated preparation and for carbachol in the preparation treated with phenoxybenzamine were significantly steeper than that for carbachol in the untreated preparation, suggesting that carbachol in the phenoxybenzamine-treated preparation and pilocarpine induced a greater tension for a given increase in low [Ca2+]i than did carbachol. Thus an activation of PrBCM-sensitive cholinoceptors might enhance the Ca2+-sensitivity of the contractile elements.  (+info)

Muscarinic acetylcholine receptors. Peptide sequencing identifies residues involved in antagonist binding and disulfide bond formation. (7/35)

Muscarinic acetylcholine receptors (mAChR) were purified from rat brain and labeled either with the site-directed affinity label [3H]propylbenzilylcholine mustard (PrBCM) or with the sulfhydryl-specific label [3H]N-ethylmaleimide (NEM), using a protocol designed to give selective incorporation of the label into disulfide-bonded cysteines. m1 mAChRs were purified from CHO-K1 cells stably expressing the cloned receptor sequence and labeled with [3H]PrBCM. The labeled receptors were cleaved with the lysine-specific protease Lys-C and, after fractionation of the products, subcleaved with cyanogen bromide. Two major CNBr cleavage products were found with a molecular mass of approximately 3.9 and approximately 2.4 kDa, labeled either by [3H]PrBCM or [3H]NEM. The results obtained from CNBr cleavage of purified forebrain receptors were consistent with those obtained from the purified cloned m1 mAChR. Edman degradation was applied to the CNBr peptides. The results were compatible with the attachment of the [3H]PrBCM label to a conserved aspartic acid residue in transmembrane helix 3 of the mAChR (corresponding to Asp-105, m1 sequence) and of [3H]NEM to a conserved cysteine residue (corresponding to Cys-98, m1 sequence). These results support the hypothesis that the cysteine residue participates in a disulfide bond on the extracellular surface of the mAChRs and related G-protein-coupled receptors, while the aspartic acid residue is involved in binding the positively charged headgroup of muscarinic antagonists.  (+info)

Effect of proteolytic cleavage on functional properties of muscarinic acetylcholine receptors in rat pancreatic and parotid acinar cells. (8/35)

Muscarinic acetylcholine receptors in isolated rat pancreatic acinar cells have an apparent Mr of 88 000, which could be decreased to 46 000 by papain, as deduced by covalent binding of the specific alkylating agent [3H]propylbenzilylcholine mustard. Muscarinic receptors on papain-treated acinar cells retained the antagonist-binding site and both high- and low-affinity binding sites for the cholinergic agonist carbachol. Similar results were observed in studies with rat parotid acinar cells, although the receptors in both control and papain-treated cells were each 10 000-15 000 Da smaller than in pancreas. Additionally, muscarinic receptors in papain-treated pancreatic acinar cells retained the ability to mediate carbachol stimulation of digestive-enzyme secretion. These results demonstrate that the characteristic binding properties of muscarinic receptors for both agonists and antagonists as well as their ability to translate agonist occupancy into a physiological response are not altered by proteolytic cleavage.  (+info)

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