mAChR Number Research Articles

Genotypic m3-Muscarinic Receptors Preferentially Inhibit M-currents in DNA-transfected NG108-15 Neuroblastoma x Glioma Hybrid Cells.

The ability of different genotypic muscarinic acetylcholine receptors (mAChR) to inhibit the neural K+-current, IM, was assessed in clones of NG108-15 mouse neuroblastoma x rat glioma cells transfected with DNA for the genomic mAChRs m1 - 4 using tight-seal, whole-cell patch clamp recording. No significant inhibition of IM was seen in native (non-transfected) cells, or in m2 or m4 DNA-transfected cells at concentrations of acetylcholine up to 1 mM or muscarine up to 100 microM. Both acetylcholine and muscarine produced complete inhibition of IM in m3 DNA-transfected cells, but only partial (50 - 60%) inhibition in m1 DNA-transfected cells at maximally effective concentrations. This difference could not be explained by differences in mAChR number, as measured by radioligand binding and was not eliminated by adding GTP to the pipette. It is concluded that genotypic m3 receptors couple most effectively to IM and that this may explain previously reported instances of pirenzepine-resistant IM-inhibition.

Effects of atropine treatment on in vitro and in vivo binding of 4-[125I]-dexetimide to central and myocardial muscarinic receptors

Upregulation of muscarinic cholinergic receptors (mAChR) after chronic atropine treatment has been described previously. The present study was designed to evaluate 4-iodine-125 dexetimide as an agent to determine changes in the number of mAChR. Rats were injected subcutaneously with atropine (500 mg/kg) either once or chronically, once daily for 10 days, and sacrificed 24 h later. In vitro binding assays with 4-[125I]-dexetimide showed significant increases in the number of mAChR in cerebra (21%) and ventricles (45%) after chronic atropine treatment but not after acute treatment. The affinity of binding to cerebral and ventricular mAChR declined after acute and chronic atropine treatment. In vivo studies were carried out involving intravenous injection of 4-[125I]-dexetimide 24 h after atropine treatment. Binding was markedly reduced in the brain and heart. Upregulation of mAChR, as seen in in vitro studies, could not be observed because of the remaining atropine. Occupancy of mAChR by atropine persisted as long as 7 days after one dose. The results of these studies indicate that 4-[125I]-dexetimide binding reflects the effects of atropine on central and peripheral muscarinic cholinergic receptors in vitro and in vivo.

Regulation by High Density Lipoproteins of Muscarinic Acetylcholine Receptor Function in Chick Heart Cells Cultured in Defined Medium

Activation of cardiac muscarinic acetylcholine receptors (mAChR) on cultured chick heart cells results in a decrease in cellular cAMP levels and a stimulation of phosphoinositide breakdown. A serum-free culture system has been used to investigate the regulation of mAChR number and function by purified serum high density lipoprotein (HDL). Administration of HDL purified from rooster serum to chick heart cells cultured in defined medium results in an attenuation of the ability of muscarinic agonist to inhibit forskolin-stimulated cAMP accumulation, with no change in its ability to stimulate phosphoinositide hydrolysis or to mediate down-regulation of receptor number. The inclusion of HDL in the culture medium did not result in appreciable changes in mAChR number or affinity, nor were the levels of the inhibitory guanine nucleotide-binding regulatory proteins (G-proteins) altered. However, the ability of guanine nucleotides to inhibit forskolin-stimulated adenylate cyclase activity was reduced by HDL treatment, suggesting that HDL interferes with the capacity of G-proteins to interact with adenylate cyclase. In order to determine which component of native HDL mediates the decreased effectiveness of carbachol, the ability of lipid and apoprotein fractions to mimic the effect of HDL was tested. HDL lipid fractions were able to mimic the effect of native HDL, while protein fractions were not. This result suggests that the ability of HDL to attenuate muscarinic receptor function is mediated by its lipid constituents. The effect of HDL and HDL lipid fractions were not correlated with changes in membrane cholesterol content.

A role for potassium channels in the regulation of cortical muscarinic acetylcholine receptors in an in vitro slice preparation

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 [ 3 H]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 °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 γ-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 Ca 2+-dependent K +-channels. Only TEA and 4-AP diminished carbachol-induced down-regulation suggesting that this effect involves only the non Ca 2+-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.

Muscarinic-agonist and Guanine Nucleotide Stimulation of Myo-Inositol Trisphosphate Formation in Membranes Isolated from Bovine Iris Sphincter Smooth Muscle: Effects of Short-Term Cholinergic Desensitization richard E. Honkanen

The effect of short-term cholinergic desensitization on muscarinic acetylcholine receptor (mAChR)-mediated activation of phospholipase C was investigated in membranes isolated from the bovine iris sphincter smooth muscle. Membranes prepared from normal or desensitized muscles, prelabeled with either [3H]myo-inositol or 32P from [gamma-32P]ATP, were incubated with a hydrolysis-resistant analogue of GTP, GTP gamma S, or GTP gamma S plus carbachol (CCh), and the production of [3H]myo-inositol 1,4,5-trisphosphate (IP3) and the breakdown of polyphosphoinositides were assessed. In normal membranes, GTP (greater than or equal to 1 mM), GTP gamma S (greater than 10 microM) and GTP gamma S (1 microM) plus CCh (10 microM), but not GDP or GDP beta S, increased phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and IP3 production. GTP gamma S increased IP3 accumulation in a time- and dose-dependent manner, and CCh, which had no effect on phospholipase C activity in the absence of GTP gamma S, potentiated the effects of GTP gamma S. The effect of CCh plus GTP gamma S on IP3 production was inhibited by atropine, had an absolute requirement for nM amounts of Ca2+ and was not affected by pertussis toxin. At higher concentrations (greater than 1 microM), Ca2+ alone induced PIP2 hydrolysis. Short-term exposure (less than 60 min) of the muscle to CCh (100 microM) did not affect the total number (Bmax) of mAChRs nor their affinity (KD) for [3H]-N-methylscopolamine. Desensitization did, however, result in: (1) a loss of the CCh-high affinity binding state of the sphincter mAChRs in a manner analogous to that produced by GTP gamma S; (2) a loss of the ability of GTP gamma S to affect CCh binding to the receptors; and (3) an attenuation of the GTP gamma S plus CCh-stimulated PIP2 hydrolysis. In conclusion, the data presented suggest that, in the iris smooth muscle, G-proteins are involved in the coupling of mAChRs to phospholipase C and that short-term cholinergic desensitization results in (1) the uncoupling of the receptor-G-protein complex and (2) the attenuation of mAChR-activation of phospholipase C.

Characterization of muscarinic acetylcholine receptors in rat cerebral cortex slices with concomitant morphological and physiological assessment of tissue viability

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 [ 3 H]N- methyl scopolamine ([ 3H]NMS) and [ 3H]quinuclidinyl benzylate ([ 3H]QNB) in this preparation. Trypan blue exclusion tests in 400-μm-thick cortical slices showed a number of dead cells in a 100 μm 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 °C, and after 3 h at 30 °C. Binding of both ligands could be displaced by unlabelled atropine sulphate, NMS or QNB. Saturation binding curves yielded a B max 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 °C. K d 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.

Alteration in the regulation of neuronal muscarinic acetylcholine receptor number induced by chronic lithium in neuroblastoma cells

Because previous studies in whole-animal models have reported inconsistent results regarding the effect of chronic lithium on the regulation of the neuronal muscarinic acetylcholine receptor (mAChR) number, we examined the effect of chronic lithium on the regulation of mAChR in cell cultures of N1E-115, a mouse neuroblastoma clone, Li + induced a concentration- and time-dependent increase in the mAChR number, with a 30% increase in specific [ 3H]quinuclidinyl binding in membrane homogenates induced by a 5-day incubation with 10 mM Li +. Agonist-induced down-regulation of the mAChR number was also inhibited by lithium: chronic treatment with 10 mM Li + caused a 25–35% reduction in the magnitude of carbachol. In contrast, the decrease in the mAChR number induced by the synergistic action of A23187 (300 nM) and phorbol myristate acetate (300 nM) was unaffected by Li +. These results demonstrate that chronic treatment with Li + increases the basal mAChR number and dampens the decrease in receptor number induced by a muscarinic agonist in neuroblastoma cells. The implications of these results in understanding the functional regulation of neuronal mAChR number are discussed.

Biochemical and physical analyses of newly synthesized muscarinic acetylcholine receptors in cultured embryonic chicken cardiac cells

Exposure of cultured embryonic chicken cardiac cells to the muscarinic agonist carbachol results in a 70-80% decrease in the number of muscarinic acetylcholine receptors (mAChR) expressed on the surface of the cells. Removal of the agonist results in a gradual increase in mAChR number because of the accumulation of newly synthesized receptors, reaching the control level in 14 hr. Measurements of increases in K+ permeability elicited by carbachol show that even after the complete recovery of receptor number, the sensitivity to agonist is reduced. The EC50 for carbachol is 13-fold higher in cells that have been exposed to carbachol and allowed to recover for 18 hr than in control cells, but is not significantly different from the EC50 for control cells 24 hr after agonist removal. The sensitivity of the mAChR-mediated inhibition of adenylate cyclase is also decreased at 18 hr, and recovers by 24 hr. These increases in sensitivity of mAChR-mediated responses are not blocked by administration of cycloheximide, and thus do not require de novo protein synthesis. The number of surface mAChR available for ligand binding can be reduced by 85-100% by treatment with the affinity-alkylating antagonist propylbenzilylcholine mustard. Newly synthesized mAChR that appear following affinity alkylation are also poorly coupled to mAChR-mediated increases in K+ permeability, indicating that decreased physiological sensitivity is not due to a nonspecific effect of long-term agonist exposure on general cellular function, but reflects, rather, an intrinsic property of newly synthesized mAChR. The decrease in sensitivity of the mAChR-mediated responses is due neither to a lack of expression of mAChR on the surface nor to reduced agonist affinity of the mAChR. Cells exhibiting decreased responsiveness contain GTP-binding proteins, which function normally in the inhibition of adenylate cyclase and appear to be identical to pertussis toxin substrates from control cells using gel electrophoresis; therefore, the decreased sensitivity does not appear to be the result of an alteration in coupling proteins. These cells also contain mAChR that do not differ from those in control cells either by molecular weight or isoelectric point. Thus, the diminished sensitivity observed in cells containing newly synthesized receptors is either caused by a small change in mAChR not detected by these electrophoretic techniques or by a change in an as-yet-undefined component of mAChR transduction system in the heart.

Activation of protein kinase C induces rapid internalization and subsequent degradation of muscarinic acetylcholine receptors in neuroblastoma cells.

The tumor-promoting phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), which activates protein kinase C, acted synergistically with A23187 to decrease muscarinic acetylcholine receptor (mAChR) number in neuroblastoma cells (clone N1E-115) as determined by a filter binding assay using [3H]quinuclidinyl benzilate in membrane homogenates. After a 6-h incubation, 10(-7) M PMA and 3 X 10(-7) M A23187 reduced mAChR number 30-40%, compared to the 40-50% reduction observed after treatment with 10(-3) M carbachol, a muscarinic agonist. Incubation with 3 X 10(-7) M A23187 and 10(-7) M 4 alpha-phorbol 12,13-didecanoate, an inactive phorbol ester, did not alter mAChR number. The addition of PMA and A23187 to cultures incubated with 10(-3) M carbachol caused only a modest 6% further reduction in mAChR number as compared to incubation with carbachol alone. The kinetics of the decrease in mAChR number produced by PMA/A23187 were similar to those seen after carbachol treatment. Recovery of mAChR number after treatment with either carbachol or PMA/A23187 was blocked by treatment with the protein synthesis inhibitor cycloheximide. Intact cell binding studies employing [3H]N-methylscopolamine showed that treatment with either PMA/A23187 or carbachol caused a rapid (within 15 min) loss of receptors from the cell surface prior to the decrease in total mAChR number. PMA (10(-7) M), but not 4 alpha-phorbol 12,13-didecanoate, promoted the translocation of protein kinase C activity from the cytosol to the membrane. Incubation with carbachol increased membrane-associated protein kinase C activity within 5 min with an EC50 of 3 X 10(-6) M. This increase persisted for at least 60 min in the continued presence of carbachol and was blocked by simultaneous incubation with atropine. These results suggest that activation of protein kinase C may be involved in the regulation of mAChR number in response to agonist.

Decreased Muscarinic Acetylcholine Receptor Number in the Central Nervous System of the Tottering (tg/tg) Mouse

The tottering mouse (tg/tg) is a single-locus mutant, phenotypically characterized by the development of epilepsy associated with distinct electroencephalographic abnormalities. Because of reported alterations in muscarinic receptor (mAChR) number in various seizure states, mAChR density was examined in discrete brain regions of tottering (tg/tg) and coisogenic wild-type (+/+) mice. Saturation binding experiments revealed a widespread decrease in membrane mAChR density in the CNS of adult tottering (tg/tg) mice as compared with age-matched control wild-type (+/+) mice. The decrease was most pronounced in the hippocampus, where tg/tg mice exhibited a 40-60% reduction in mAChR density with no change in the affinity of the receptor for antagonists or agonists. At postnatal day 10, before the reported onset of electroencephalographic abnormalities, 114 and 65% increases in mAChR density were observed in the tg/tg hippocampus and cortex, respectively. Following the development of seizure activity at postnatal day 22, mAChR density in the tg/tg hippocampus was reduced by 29%. No change in brain mAChR density was seen in adult heterozygotes (+/tg), which do not develop electroencephalographic or seizure abnormalities. These results indicate that the development of reduced mAChR number in the CNS of the tg/tg mouse is secondary to abnormal neuronal activity, providing further support for the hypothesis that membrane depolarization can cause a decrease in neuronal mAChR density.

Regulation of Neuronal Muscarinic Acetylcholine Receptor Number by Protein Glycosylation

Tunicamycin, a potent inhibitor of protein glycosylation, was used to study the role of protein glycosylation in the regulation of muscarinic acetylcholine receptor (mAChR) number in cultures of N1E-115, a murine neuroblastoma cell line. At a concentration of 0.35 microgram/ml, tunicamycin inhibited macromolecular incorporation of [3H]mannose by 75-80%, whereas incorporation of [3H]leucine was reduced by only 10%. Treatment with tunicamycin caused a 30% decrease in total membrane mAChR number within 48 h as determined by a filter-binding assay using [3H]quinuclidinyl benzilate ([3H]QNB), a highly specific muscarinic antagonist. Tunicamycin also inhibited the recovery of total membrane mAChR by 70% following carbachol-induced down-regulation. The rate of mAChR degradation (control t1/2 12-14 h) was unaffected by incubation with tunicamycin. Intact cell binding studies using [3H]QNB (a membrane-permeable ligand) to measure total cellular (internal plus cell surface) mAChR and [3H]N-methylscopolamine ([3H]NMS, a membrane-impermeable ligand) to measure cell surface mAChR were conducted to determine whether tunicamycin selectively depleted cell surface mAChR. With 12 h of treatment with tunicamycin, cell surface mAChR number declined by 35%, whereas total cellular mAChR fell by only 10%. The ratio of cell surface receptor to total receptor decreased by 45% after 24 h. These results indicate that protein glycosylation is required for the maintenance of cell surface mAChR number. Incubation with tunicamycin causes a selective depletion of cell surface mAChR, implying that protein glycosylation plays a critical role in transport and/or incorporation of mAChR into the plasma membrane.

Reduction of muscarinic acetylcholine receptor number and affinity by an endogenous substance.

We examined the soluble fraction from homogenates of 12-day embryonic chick heart for the presence of an endogenous modulator of muscarinic acetylcholine receptors (mAChR). Homogenates were separated into 100,000 g soluble and crude membrane fractions by differential centrifugation. Aliquots of membranes were incubated in the presence or absence of the soluble fraction and the muscarinic antagonist, [3H]quinuclidinyl benzilate ( [3H]QNB), and the data subjected to Scatchard analysis. In the presence of the soluble fraction, mAChR number decreased up to 70% and the affinity for [3H]QNB decreased six- to eightfold. These results suggested that an endogenous soluble factor (ESF) affected cholinergic ligand binding to the receptor. The amount of ESF extracted from less than 10 mg of brain was sufficient to reduce by 50% [3H]QNB binding to 50 fmol mAChR. ESF activity was partially purified by heat and acid treatment. The loss of receptors was dependent upon the amount of ESF added and was time dependent. QNB protected some receptors from loss due to ESF. The change in mAChR affinity for [3H]QNB was observed only if ESF was present continuously during the [3H]QNB binding assay. Ultrafiltration and gel filtration showed that ESF was less than 10,000 daltons and probably less than 700 daltons. ESF activity was blocked by EDTA. However, ESF was not a divalent cation since it was base labile, and removal of divalent cations with Chelex-100 did not inhibit ESF activity. ESF activity was also blocked by catechol, catecholamines, ascorbate, and dithiothreitol. ESF was present in embryonic but not in adult heart.

Seasonal variations in the muscarinic regulation of acetylcholine release from torpedo electric organ nerve terminals

1. 1. The cholinergic nerve endings of the electric organs of Torpedo ocellata contain presynaptic muscarinic acetylcholine receptors (mAChR) which regulate acetylcholine (ACh) release by negative feedback. 2. 2. The efficiency of this muscarinic regulation varies circannually: maximal inhibition is observed in the winter, much smaller effects in the fall and spring, and no effect is observed during the summer. 3. 3. These variations are accompanied by seasonal changes in the ability of the mAChR to trigger the synthesis of its second messenger (a prostaglandin E-like substance) and in the ability of exogenous prostaglandin E 2 to inhibit ACh release. No seasonal changes were found in the number of presynaptic mAChRs. 4. 4. These findings suggest that the observed seasonal variations are due to changes in both the metabolism of prostaglandins in the electric organ and the sensitivity of the ACh-releasing apparatus to the muscarinic second messenger.