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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This work explored the role of the cholinergic pathway, assessed at a post-synaptic level by the use of isolated smooth muscle cells, in the impairment of antral motility associated with diabetic gastroparesis. Contractile response to carbachol--but not to erythromycin, a motilin receptor agonist--was abolished in antral smooth muscle cells isolated from (i) rats previously rendered diabetic by a single i.v. dose of streptozotocin (STZ, 60 mg/kg) and (ii) db/db spontaneously diabetic mice. Insulin treatment of STZ-rats was able to prevent the impairment of the carbachol contractile response, but not to reverse it once established. In STZ-rats, impairment of contractile response was not associated with a change in density of [3H]-N-methyl-scopolamine ([3H]-NMS) binding sites (approximately 1.5 fmol/mg protein). Displacement curve of the [3H]-NMS binding by carbachol was shifted to the right in diabetic rats as compared to controls. The addition of GTP-gamma-S induced a shift to the right of the displacement curve in control but not in diabetic animals. These results strongly suggest that diabetes is associated with an early and specific alteration of the muscarinic control of contraction of antral smooth muscles at a post-synaptic level, associated with an alteration of the GTP-binding proteins coupled to muscarinic receptors.
Mol Cell Biochem 1992 Feb 12
PMID:Impairment of contractile response to carbachol and muscarinic receptor coupling in gastric antral smooth muscle cells isolated from diabetic streptozotocin-treated rats and db/db mice. 138 42

A large number of diverse pharmacological agents bind to a secondary domain on the muscarinic receptor, to influence allosterically the interaction of ligands at the primary binding site. Based on common structural features of these antagonists, we examined the interaction of protamine, an endogenous polycationic peptide, and of polyamines with muscarinic receptors in rat heart. Our results provide several lines of qualitative evidence that protamine allosterically modulates the conformation of muscarinic receptors, in a marked negatively cooperative manner. It decelerated the dissociation of N-[3H]methylscopolamine ([3H] NMS) initiated by atropine, in a concentration-dependent fashion. Inhibition by protamine of [3H]NMS binding at equilibrium showed a distinct plateau, which increased in magnitude at higher ligand concentrations. Scatchard analysis of saturation isotherms of [3H]NMS binding in the absence and presence of protamine indicated that protamine did not alter Bmax in a statistically significant fashion, although there was a trend of a concentration-dependent increase in this parameter. On the other hand, it caused a marked concentration-dependent decrease in the affinity of [3H]NMS, and this effect reached a ceiling limit. However, there were marked quantitative deviations of the interaction of protamine from a simple ternary allosteric model. Some of these discrepancies could be explained by the tendency of protamine to increase Bmax. The allosteric actions of protamine demonstrated in kinetic and equilibrium experiments were selective for m1 and m2 muscarinic receptors, compared with m3, m4, and m5 receptors, as studied in Chinese hamster ovary cells transfected with the genes of the different muscarinic receptors. Arginine residues play an important role in the allosteric interaction of protamine, inasmuch as poly-L-arginine qualitatively mimicked the effects of protamine. In contrast, no effects of the polyamines spermine, spermidine, and putrescine were observed on [3H]NMS binding. This is the first report on the allosteric modulation of muscarinic receptors by an endogenous peptide.
Mol Pharmacol 1992 Aug
PMID:Complex allosteric modulation of cardiac muscarinic receptors by protamine: potential model for putative endogenous ligands. 151 29

The role of sialic acid residues in the interactions of muscarinic agonists with the cardiac M2 muscarinic receptor was investigated by competitive binding experiments using the lipophilic radioligand (-)-[benzilic-4,4-3H]quinuclidinyl benzilate ([3H]QNB) and the hydrophilic ligand [N-methyl-3H]scopolamine methyl chloride ([3H]NMS). Direct labeling of the agonist binding sites was performed with the radiolabeled agonist [methyl-3H]oxotremorine M acetate ([3H]oxo-M). Neuraminidase decreased the affinity of the M2-selective agonist carbamylcholine in competitive binding experiments performed with [3H]QNB and [3H]NMS. The binding of the M1-selective agonist (4hydroxy-2-butynyl)trimethylammonium chloride m-chlorocarbanilate (McN-A-343), of the M1-selective antagonist pirenzepine, and of the M2-selective antagonist 11-([2-[(diethylamino)methyl]-1 piperidinyl]acetyl)-5,11-dihydro-6H-pyrido(2,3b)(1,4)benzodiazepin -6-on (AF-DX-116) were not affected by neuraminidase. Neuraminidase did not modify the binding parameters of 3H-antagonists but reduced the number of agonist binding sites revealed by [3H]oxo-M. The removal of sialic acid decreased the half-life of the receptor-agonist complex. The present results suggest that removal of sialic acid reduces the formation of super-high affinity agonist-receptor complexes. Sialic acid may catalyze macroscopic binding by enhancing accumulation of the agonist at the membrane surface.
Mol Pharmacol 1990 May
PMID:Sialic acid residues as catalysts for M2-muscarinic agonist-receptor interactions. 169 6

The effect of the neuromuscular blocker alcuronium on the binding of N-[3H]-methylscopolamine [( 3H]NMS) and l-[3H]quinuclidinylbenzilate ([3H]QNB) to muscarinic binding sites in rat heart atria, longitudinal smooth muscle of the ileum, cerebral cortex, cerebellum, and submaxillary glands was measured using filtration techniques. In the presence of 10(-5) M alcuronium, the binding of [3H]NMS (which was present at a subsaturating concentration of 2 x 10(-10) M) was increased 5.3-fold in the atria and smooth muscle and 3-fold in the cerebellum; no increase was observed in the brain cortex and salivary glands. The binding of [3H]NMS was inhibited at 10(-3) M and higher concentrations of alcuronium. The rates of [3H]NMS association to and dissociation from muscarinic binding sites in the atria were diminished by 10(-5) M alcuronium. Scatchard plots of [3H]NMS binding data obtained with and without 10(-5) M alcuronium indicated that the maximum number of binding sites was not altered by the drug, whereas the apparent Kd for [3H]NMS was diminished. In contrast to [3H] NMS, the effects of alcuronium on the binding of [3H]QNB were only inhibitory. The concentration of alcuronium required to diminish the binding of [3H]QNB by 50% (IC50) was 4-7 microM in the atria, ileal smooth muscle, and the cerebellum, 140 microM in the brain cortex, and 1200 microM in the parotid gland. The results suggest that the binding of low concentrations of alcuronium to muscarinic receptors in the heart, ileal smooth muscle, and cerebellum allosterically increases the affinity of muscarinic receptors towards [3H]NMS, although not [3H]QNB. At high concentrations, alcuronium inhibits the binding of muscarinic ligands, presumably by competition for the classical muscarinic binding site. Positive cooperativity induced by alcuronium appears to be specific for the m2 (cardiac) subtype of muscarinic receptors.
Mol Pharmacol 1990 Nov
PMID:Positive cooperativity in the binding of alcuronium and N-methylscopolamine to muscarinic acetylcholine receptors. 223

Cell surface muscarinic cholinergic receptors have been characterized and quantified for the first time, in intact, isolated adult rat cardiomyocytes. The cells were previously established as functionally fully compatible with cellular responses in intact cardiac tissue. The specific binding of the hydrophilic radioligand, [3H]-NMS, (N-methyl-[3H]-scopolamine methylchloride) was found to be stereo-specific, saturable, reversible and of high affinity. Binding of [3H]-NMS demonstrated appropriate drug specificity and was positively correlated with increasing cell concentrations. Bmax for [3H]-NMS binding to ventricular myocytes, enzymatically dissociated from adult male rats, was 15.8 +/- 1.03 fmol/25 x 10(3) cells (at 4 degrees C) and KD was 0.27 +/- 0.05 nM (n = 14). Binding assays performed at a higher incubation temperature (30 degrees C) yielded a higher Bmax value (22.1 +/- 1.6 fmol/25 x 10(3) cells; n = 11; P less than 0.005 vs. Bmax at 4 degrees C) but an unchanged KD (0.23 +/- 0.06 nM). Pretreatment of myocytes with the muscarinic agonist carbachol (1 mM) at 37 degrees C resulted in a reduction (down-regulation) in specific binding of the hydrophilic ligand [3H]-NMS. The magnitude of this reduction and its rate of recovery were dependent on the time of the exposure to carbachol. Exposures of 30-60 min elicited down-regulated by 35% (Bmax = 14.29 +/- 1.66 changed to 9.5 +/- 1.79 fmol/25 x 10(3) cells, without change in KD P less than 0.01, n = 4). The down-regulation of the muscarinic receptors by carbachol was insensitive to application of bacitracin - an inhibitor of endocytosis. On the other hand preincubation with 10(-9)M atropine, a muscarinic antagonist, hindered the agonist-induced receptor "loss" from the cell surface confirming the muscarinic nature of these receptors. We conclude that our preparation of intact, isolated ventricular cardiomyocytes is ideally suited for the study of cell surface muscarinic receptor regulation under physiological and pathological conditions.
J Mol Cell Cardiol 1990 Nov
PMID:Characterization and agonist regulation of muscarinic ([3H]N-methyl scopolamine) receptors in isolated ventricular myocytes from rat. 228 85

Some novel observations dealing with antagonist binding to cardiac particulate muscarinic receptors are described. Gpp(NH)p increased (2-3 fold) the specific binding of [3H]-QNB or [3H]-NMS, both potent muscarinic antagonists, to washed particles (WP), but not microsomes (MIC), when the binding was conducted at 30 degrees C. Magnesium, on the other hand, increased (2-3 fold) the binding of these antagonists to MIC, but not to WP, under the same condition. The treatment of subcellular fractions with 0.2 mM N-ethylmaleimide (NEM), a sulfhydryl reagent, failed to significantly modify the respective stimulatory actions of either Gpp(NH)p on WP binding or of magnesium on MIC binding of these antagonists; treatment with dithiothreitol (1 mM) was also ineffective in this regard. Gpp(NH)p decreased Kd (WP) while magnesium increased Kd (MIC) for [3H]-QNB. Repeated freezing/thawing of isolated subcellular fractions abolished the stimulatory effect of magnesium on antagonist binding to MIC but not of Gpp(NH)p on WP antagonist binding; the freeze/thaw procedure per se increased MIC binding but not WP binding of these antagonists. When the binding was conducted at 4 degrees C (24 hr), the stimulatory effect of Gpp(NH)p on [3H]-QNB binding was enhanced (6-fold) in the case of WP and was detectable (80%) in the case of MIC. Under this condition, the stimulatory effect of magnesium on [3H]-QNB binding was also enhanced (5-fold) in the case of MIC and became evident (200%) in the case of WP. The results of this work support the following views: (a) antagonist-occupied cardiac muscarinic receptors are capable of interaction with guanine nucleotide binding proteins (G protein like Gi, Go) and such interaction influences antagonist binding properties (e.g. increased affinity) of the cardiac membrane-associated muscarinic receptors (b) magnesium influences (decreased affinity) antagonist binding properties by interacting with multiple sites of which some are likely associated with components other than G proteins of the particulate fractions (c) a pool of NEM-sensitive sulfhydryls involved in the regulation of Gpp(NH)p-sensitive agonist binding to cardiac muscarinic receptors is not involved in the regulation by either Gpp(NH)p or magnesium of antagonist binding in these subcellular fractions and (d) membrane fluidity and microenvironment surrounding the receptor and G proteins contribute to the actions of Gpp(NH)p and magnesium on antagonist binding.
Mol Cell Biochem 1990 May 10
PMID:MgCl2-sensitive and Gpp(NH)p-sensitive antagonist binding states of rat heart muscarinic receptors: preferential detection at ambient temperature assay and location in two subcellular fractions. 237 47

To compare the proportions of four muscarinic receptors in different rat brain regions, we used competition curves with four selective antagonists, at 1-[N-methyl-3H]scopolamine methyl chloride [( 3H]NMS) binding equilibrium and after allowing [3H]NMS dissociation for 35 min. Himbacine and methoctramine were shown to discriminate two muscarinic receptor subtypes having a high affinity for 4-diphenylacetoxy-N-methylpiperidine methiodide and hexahydrosiladifenidol, intermediate affinity for pirenzepine, and low affinity for AF-DX 116. One M4 subtype had a high affinity for himbacine and methoctramine; it was found predominantly in homogenates from rat striatum (46% of total [3H]NMS receptors) and in lower proportion in cortex (33% of [3H]NMS receptors) and hippocampus (16% of [3H]NMS receptors). Its binding properties were identical to those of muscarinic receptors in the neuroblastoma x glioma NG 108-15 hybrid, suggesting that it was encoded by m4 mRNA. The M3 subtype (typically found in rat pancreas, a tissue expressing the m3 mRNA) had a low affinity for himbacine and methoctramine and represented about 10% of all [3H]NMS receptors in rat brain cortex, hippocampus, striatum, and cerebellum. M1 and M2 receptors were identified in rat brain by their high affinity for pirenzepine and AF-DX 116, respectively.
Mol Pharmacol 1990 Aug
PMID:Binding of selective antagonists to four muscarinic receptors (M1 to M4) in rat forebrain. 238 34

The rules underlying muscarinic acetylcholine receptor (mAChR) regulation in an in vitro cortical slice preparation of adult rats were examined following various alterations of bioelectric activity and following agonist stimulation. Muscarinic ACh antagonists [3H]N-methyl scopolamine ([3H]NMS) or [3H]quinuclidinyl benzylate ([3H]QNB) were used to label cell surface vs total (i.e. surface and internal) receptors, respectively. Depolarization of neural membranes for 4 h at 22-37 degrees C using veratridine or high external potassium (K+) led to a temperature-dependent down-regulation of surface mAChR of 26.2% and 11.3%. Total mAChRs decreased by 37.6% and 8.1%. Addition of picrotoxin and glutamic acid also led to decreases in mAChRs. Increases in inward chloride ion current induced by gamma-aminobutyric acid (GABA) or gold chloride had no significant effect on mAChRs. Blockade of calcium channels and synaptic transmission by magnesium or cobalt and postsynaptic calcium channels with nifedipine showed a significant effect on mAChRs only in the latter case. In contrast, agonist stimulation using carbachol led to a large down-regulation for both [3H]NMS and [3H]QNB (26.1%, 35.9%). ACh decreased [3H]QNB binding by 33.9%, but had little effect on [3H]NMS binding (6.3%). For [3H]QNB binding sites the effects of carbachol appeared to summate with those of veratridine. Down-regulation of [3H]NMS labelled mAChRs by carbachol and veratridine had an estimated half-time of 30 min and 2 h, respectively. Neither the effects of veratridine nor carbachol could be antagonized by tetrodotoxin (TTX), showing that the effects were not due to an increase in sodium ion currents. However, a common thread linking the various agents which induce mAChR down-regulation appears to involve changes in potassium (K+) current. Potassium channel blockers tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP) and apamin had little independent effect on mAChR number, but prevented veratridine-induced down-regulation, presumably through a blockade of K+- and Ca2+-dependent K+-channels. Only TEA and 4-AP diminished carbachol-induced down-regulation suggesting that this effect involves only the non Ca2+-dependent K+-channels. It thus appears that mAChR regulation in the rat cerebral cortex is linked to changes in active K+-channel currents: activation of the K+-channel by depolarization-induced changes in K+ current or by agonist stimulation leading to changes in the selective K+ currents stimulate mAChR down-regulation; blockage of the K+-channels prevents this down-regulation.
Brain Res Mol Brain Res 1989 Jan
PMID:A role for potassium channels in the regulation of cortical muscarinic acetylcholine receptors in an in vitro slice preparation. 253 5

Stimulation of muscarinic acetylcholine receptors (mAChR) elicits phosphatidylinositol turnover, which yields inositol phosphates (InsP) and diacylglycerol (DG) the latter activating protein kinase C (PKC). Activating PKC with phorbol esters inhibits mAChR agonist-stimulated phosphoinositide hydrolysis and InsP production. A possible mechanism of this inhibition may be down-regulation of mAChR by PKC. In the present work, rat cortical slices were preincubated with phorbol 12,13-dibutyrate (PDBu) followed by binding assays for [3H]quinuclidinyl benzilate [( 3H]QNB), [N-methyl-3H]scopolamine [( 3H]NMS) or [3H]pirenzepine [( 3H]PZ). Our data demonstrate that activation of PKC by phorbol esters causes a rapid down-regulation of muscarinic cholinergic receptors. This down-regulation is also rapidly reversible. Receptors on the cell surface appear to be more sensitive to the effect of PKC than do internal ones. This down-regulation occurs by a decrease in the number of receptors, rather than by changes in receptor affinity. The results suggest that PKC may exert negative feedback on its own activation by down-regulating the receptors that normally elicit phosphatidylinositol turnover.
Brain Res Mol Brain Res 1989 Jun
PMID:Phorbol 12,13-dibutyrate regulates muscarinic receptors in rat cerebral cortical slices by activating protein kinase C. 274 52

We have begun studies on regulatory mechanisms of muscarinic acetylcholine receptors (mAChRs) in slices of rat cerebral cortex. This paper, the first of two, deals with the viability of the cells in the slices (a prerequisite for studying receptor regulation) and provides a characterization of binding sites for [3H]N-methyl scopolamine ([3H]NMS) and [3H]quinuclidinyl benzylate ([3H]QNB) in this preparation. Trypan blue exclusion tests in 400-microns-thick cortical slices showed a number of dead cells in a 100 microns zone from each cut edge, for a total of about 15-30% of all cells in the slice. In agreement with previous reports, electron microscopy revealed healthy tissue in the middle of the slice, but after incubation for several hours, swollen cells and dendrites were seen without cytoplasmic organelles. Axon terminals, however, were still seen to synapse upon these processes. Electrophysiological single unit recordings showed spontaneous action potentials in the slices. For receptor binding experiments, slices were incubated with either [3H]NMS, a hydrophilic mAChR ligand which does not penetrate the cell membrane, or the lipophilic ligand [3H]QNB which readily enters cells. For both ligands, equilibrium binding was reached after 8 h at 4 degrees C, and after 3 h at 30 degrees C. Binding of both ligands could be displaced by unlabelled atropine sulphate, NMS or QNB. Saturation binding curves yielded a Bmax of 2187 fmol/mg protein for [3H]QNB (reflecting all mAChRs) and 1335 fmol/mg protein for [3H]NMS (only mAChRs on the cell surface) at 30 degrees C. Kd values were 8.2 and 5.2 nM for [3H]QNB and [3H]NMS, respectively. These values are high compared with values obtained from homogenates, frozen sections or dissociated cells, and presumably reflect the use of intact, living tissue. These data are probably a better reflection of the actual, in vivo mAChR number and affinity than those obtained from dead tissue. This slice preparation suggests itself as a simple but effective method with which to study the regulation of mAChRs in living brain tissue.
Brain Res Mol Brain Res 1989 Jan
PMID:Characterization of muscarinic acetylcholine receptors in rat cerebral cortex slices with concomitant morphological and physiological assessment of tissue viability. 292 84


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