Regulation Of Muscarinic Acetylcholine Receptors Research Articles

Hippocampus-dependent fear conditioning is not sensitized by muscarinic receptor activation following systemic injection of pilocarpine

The regulation of muscarinic acetylcholine receptors (mAChR) critically influences emotional outcomes. Previous researches indicate that a single systemic injection of pilocarpine – a mAChR agonist – displays long-term defensive behaviors in rats evaluated in distinct unconditioned tests up to 3 months following treatment. However, it is not clear whether these effects share underlying behavioral phenotypes involved in conditioned responses. With this in mind, we examined whether mAChR activation modulates contextual fear conditioning (CFC) and/or hippocampal synaptic plasticity. Adult male Wistar rats were injected with pilocarpine (150 mg/kg) and behaviorally evaluated in the CFC test or followed by synaptic plasticity (LTP/LTD) investigation in CA1 stratum radiatum of hippocampal slices. There was no difference between groups in the quantification of freezing behavior during the test period (24 h after treatment) besides a decrease of freezing 1 month later. Similarly, no changes were observed in rats conditioned 24 h later and tested 1 month after. Synaptic plasticity investigation following short- or long-term treatment revealed no differences between control and treated subjects. In summary, our results show that hippocampus-dependent fear behavior and memory consolidation mediated by hippocampal cholinergic inputs are not sensitive to activation of mAChR by a systemic nonconvulsant dose of pilocarpine. Therefore, we suggest that the long-term defensive behaviors and anxiogenic-like features displayed by pilocarpine observed in rats are mediated by different underlying mechanisms and or set of synapses.

Conformational Complexity and Dynamics in a Muscarinic Receptor Revealed by NMR Spectroscopy.

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical GPCR that plays important roles in regulating heart rate and CNS functions. Crystal structures provide snapshots of the M2R in inactive and active states, but the allosteric link between the ligand binding pocket and cytoplasmic surface remains poorly understood. Here we used solution NMR to examine the structure and dynamics of the M2R labeled with 13CH3-ε-methionine upon binding to various orthosteric and allosteric ligands having a range of efficacy for both G protein activation and arrestin recruitment. We observed ligand-specific changes in the NMR spectra of 13CH3-ε-methionine probes in the M2R extracellular domain, transmembrane core, and cytoplasmic surface, allowing us to correlate ligand structure with changes in receptor structure and dynamics. We show that the M2R has a complex energy landscape in which ligands with different efficacy profiles stabilize distinct receptor conformations.

Capacitative Ca2+ entry is involved in regulating soluble amyloid precursor protein (sAPPα) release mediated by muscarinic acetylcholine receptor activation in neuroblastoma SH‐SY5Y cells

Previous studies have demonstrated that stimulation of phospholipase C-linked G-protein-coupled receptors, including muscarinic M1 and M3 receptors, increases the release of the soluble form of amyloid precursor protein (sAPPalpha) by alpha-secretase cleavage. In this study, we examined the involvement of capacitative Ca2+ entry (CCE) in the regulation of muscarinic acetylcholine receptor (mAChR)-dependent sAPPalpha release in neuroblastoma SH-SY5Y cells expressing abundant M3 mAChRs. The sAPPalpha release stimulated by mAChR activation was abolished by EGTA, an extracellular Ca2+ chelator, which abolished mAChR-mediated Ca2+ influx without affecting Ca2+ mobilization from intracellular stores. However, mAChR-mediated sAPPalpha release was not inhibited by thapsigargin, which increases basal [Ca2+]i by depletion of Ca2+ from intracellular stores. While these results indicate that the mAChR-mediated increase in sAPPalpha release is regulated largely by Ca2+ influx rather than by Ca2+ mobilization from intracellular stores, we further investigated the Ca2+ entry mechanisms regulating this phenomenon. CCE inhibitors such as Gd3+, SKF96365, and 2-aminoethoxydiphenyl borane (2-APB), dose dependently reduced both Ca2+ influx and sAPPalpha release stimulated by mAChR activation, whereas inhibition of voltage-dependent Ca2+ channels, Na+/Ca2+ exchangers, or Na+-pumps was without effect. These results indicate that CCE plays an important role in the mAChR-mediated release of sAPPalpha.

Chapter 14 Regulation of muscarinic acetylcholine receptor expression and function

This chapter focuses on the regulation of muscarinic acetylcholine receptors (mAChR) expression and function when expressed from endogenous genes in cardiac cells and when expressed from heterologous genes in transfected cells in culture. Muscarinic receptors play a key role in the functioning of the central nervous system, where they have been implicated in such processes as memory, learning, and control of movement. Muscarinic receptors also modulate synaptic transmission in the ganglia of the autonomic nervous system and play a major role in regulating the functions of the target organs of the parasympathetic nervous system. The chapter describes several techniques of molecular and cellular biology to identify the factors that regulate the expression and function of mAChR in order to understand how cells regulate their ability to participate in cholinergic synaptic transmission. The regulation of expression of a luciferase reporter gene under the transcriptional control of a CAMP-regulated promoter is used to study the functional specificity of mAChR in transiently transfected cells.

Effects of GTP gamma S on muscarinic receptor-stimulated inositol phospholipid hydrolysis in permeabilized smooth muscle from the small intestine.

1. Smooth muscle fragments from the longitudinal layer of the small intestine of the guinea-pig were permeabilized with Staphylococcus aureus alpha toxin (alpha-toxin) and used to investigate the role of G-protein activation in the regulation of muscarinic acetylcholine receptor (AChR)-stimulated inositol phospholipid hydrolysis. 2. The efficiency of alpha-toxin permeabilization was estimated by the release of [3H]-2-deoxyglucose ([3H]-2DG) after prior loading or lactate dehydrogenase (LDH) enzyme release from the smooth muscle fragments. 3. In alpha-toxin-permeabilized smooth muscle, but not in non-permeabilized muscle, GTP gamma S induced time- and concentration-dependent increases in labelled inositol phosphates. Carbachol (CCh) increased labelled inositol phosphates in both permeabilized and non-permeabilized muscle, although the increases were greater in non-permeabilized smooth muscle. The response to 100 microM CCh was severely reduced by 0.5 microM atropine. 4. In permeabilized muscle the effects of GTP gamma S or CCh on inositol phosphate levels were reduced by treatment with pertussis toxin (PTX) and completely inhibited by GDP beta S. 5. GTP gamma S caused a concentration-dependent inhibition of the CCh-induced increases in the levels of labelled inositol phosphates. Dibutyryl cyclic AMP or Sp-cAMPs (adenosine-3',5'-cyclic phosphorothiolate-Sp) reduced the effects of CCh on inositol phosphate levels. 6. The results suggest that muscarinic AChR activation induces inositol phospholipid hydrolysis via more than one G-protein in this smooth muscle and that several mechanisms may contribute to the modulation of both stimulatory and inhibitory responses observed.

Regulation of muscarinic receptor expression by changes in mRNA stability.

Regulation of muscarinic acetylcholine receptor (mAChR) subtype mRNAs was investigated in the human neuroblastoma cell line IMR-32 and in transfected CHO cells. IMR-32 cells express both m1 and m3 subtypes of mAChR. Exposure of IMR-32 cells to the muscarinic agonist, carbamylcholine (CBC) leads to a time dependent down-regulation of mAChRs which was maximal by 9 hours. mAChR activation resulted in a differential regulation of mAChR subtype mRNAs. m1 mAChR mRNA was down-regulated following 12 hours of agonist treatment and was associated with a decreased stability of the receptor transcript. In contrast, the m3 mAChR mRNA was resistant to agonist treatment for up to 24 hours. Using transfected CHO cells, we identified sequence elements within the 3'-untranslated region (3'-UTR) of the m1 mAChR gene which dictate agonist-induced destabilization of the m1 mAChR mRNA. Removal of these sequences abolished the ability of chronic agonist exposure to destabilize m1 mAChR mRNA. These findings suggest that sequence specific differences between m1 and m3 mAChR subtypes, which both preferentially couple to hydrolysis of phosphoinositides, may be responsible for differences in the regulation of mAChR gene expression.

Clonidine prevents the short-term down regulation of muscarinic receptors in mouse brain induced by the acetylcholinesterase inhibitor soman

The influence of clonidine on the down regulation of muscarinic acetylcholine receptors induced by the irreversible acetylcholinesterase inhibitor soman was studied in mouse brain (cortex and brainstem). Muscarinic receptor binding was measured using [ N-methyl- 3H]scopolamine ([ 3H]MS) as the probe in a filtration assay. Thirty min after subcutaneous injection of soman at a dose which killed 80–90% of the mice, the concentration of [ 3H]MS binding sites was reduced by 25–28% in surviving mice. [ 3H]MS binding affinity ( K d = 0.36−0.42 nM), agonist binding state, and receptor coupling to guanine nucleotide-dependent transducer proteins (G proteins) were not affected by soman. Administration of clonidine (1 mg/kg, s.c.) 5 min prior to soman (1) prevented the decrease in receptor number and (2) decreased the extent of acetylcholinesterase inhibition caused by the soman. It is possible that a reversible inhibition of acetylcholinesterase by clonidine protects the enzyme from irreversible inactivation by soman, thereby decreasing the extent of chronic cholinergic overstimulation with its attendant down regulation of muscarinic receptors.

Regulation of muscarinic acetylcholine receptors in cultured guinea pig pancreatic acini

Regulation of muscarinic receptors in cultured guinea pig pancreatic acini was investigated by assessing the effects of cholinergic agonists on binding of [N-methyl-3H]scopolamine [( 3H]NMS) and on amylase release. Freshly dispersed acini bound [3H]NMS with a Kd of 74 pM and a maximal binding level (Bmax) of 908 fmol/mg DNA. Carbachol (CCh) stimulated amylase secretion and inhibited [3H]NMS binding. Incubation of acini for 30 min with 0.1 mM CCh decreased the subsequent efficacy of CCh in stimulating amylase release by threefold but had no effect on its potency. In contrast, amylase release in response to cholecystokinin octapeptide (CCK-8) was not altered by CCh preincubation. [3H]NMS binding to acini was decreased only 15-20% after 30-min incubation with CCh. However, culture of acini with 0.1 mM CCh decreased [3H]NMS binding by 50% at 3-4 h and by 85-90% at 24 h. This decrease was attributable primarily to a reduction in Bmax. [3H]NMS binding also was decreased to a similar extent by the cholinergic agonists bethanechol and methacholine but not by other secretagogues. The decrease in antagonist binding induced by CCh was dose dependent, with the IC50, 5.8 microM, approximating the EC50 for amylase release, 4.3 microM. Culture of acini for 24 h with CCh abolished subsequent amylase release in response to CCh but not to CCK-8. When CCh was removed from the culture medium after 24 h and acini recultured in its absence, [3H]NMS binding increased with a half-time for recovery of 20-24 h; this recovery was blocked by cycloheximide.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Use of intact rat brain cells as a model to study regulation of muscarinic acetylcholine receptors

Intact rat brain cells were dissociated and used to study the regulation of muscarinic acetylcholine receptors upon exposure to muscarinic receptor agonists. Incubation of cells with carbamylcholine resulted in a time-dependent decrease in subsequent [ 3H]N-methylscopolamine specific binding, an effect which reached a steady state after 3 hr at 37°C. This effect of carbamylcholine was dependent on the concentration of the agonist in the incubation medium and was due to a reduction in the maximal binding capacity of the receptor with no decrease in the affinity of the remaining receptors. This preparation might be useful in future studies to elucidate the mechanisms underlying the regulation of muscarinic acetylcholine receptors in the central nervous system.

Agonist Regulation of Muscarinic Acetylcholine Receptors in Rat Spinal Cord

In vitro studies with cultured cells originating from nervous tissue have shown that chronic exposure to muscarinic agonists results in a loss of muscarinic receptors. To determine whether this type of regulation of muscarinic receptor number also occurs in vivo, we infused carbachol into the spinal cords of rats. A single carbachol injection into the lumbar spinal cord caused a significant increase in the nociceptive threshold. This effect of carbachol diminished to control levels after 12 h of repeated agonist injections every 4 h and was blocked by atropine. The desensitization to the antinociceptive effects of carbachol was associated with a loss of muscarinic receptors as determined by the binding of the muscarinic antagonist [3H]quinuclidinyl benzilate. After a 24-h exposure to carbachol given every 4h, there was about a 60% loss of binding sites. The loss of muscarinic receptors was also blocked by atropine and was reversible. These results represent direct evidence that a muscarinic agonist can regulate receptor number in the central nervous system and suggest that this loss of receptors is associated with a desensitization to the antinociceptive effects of carbachol injected into the spinal cord.

Muscarinic acetylcholine receptor regulation and protein phosphorylation in primary cultures of rat cerebellum

Exposure of primary cell cultures of rat cerebellum to the muscarinic agonist carbachol led to a 50% loss in apparent muscarinic receptor number detectable in a ligand binding assay using [ 3H]quinuclidinyl benzilate ([ 3H]QNB). The loss in [ 3H]QNB binding was time-dependent with a half-life of 2.9 h. Examination of membrane protein phosphorylation indicated that exposure to carabachol resulted in an increase in the level of phosphorylation of 6 polypeptides. The increased phosphorylation of 3 of these polypeptides, of molecular weights, 75,000, 67,000 and 62,000, followed a similar time-course to that of carbachol-induced loss of receptor binding. These results are consistent with the idea that the muscarinic receptor regulation involves a protein phosphorylation mechanism.

Regulation of muscarinic acetylcholine receptors and contractility of guinea pig vas deferens

The dynamic relationships between the muscarinic acetylcholine (m-ACh) receptor and the contractility of guinea pig vas deferens were studied by measuring the density of m-ACh receptors and contractile responses to ACh and high K + (40 mM) under various experimental conditions. The guinea pig vas deferens has few spare muscarinic receptots for ACh. Brief treatment with dibenamine, an alkylating agent, decreases both the maximal contractile response to ACh and the number of m-ACh receptors to the same extent. The contractility of vas deferens, cultured for 3 days under a load of 320–1200 mg/muscle, was similar to that before culture. Addition of ACh to the culture medium resulted in a decrease in the sensitivity of the vas deferens to ACh and the number of muscarinic receptors in the muscle but did not affect noradrenaline-sensitivity, the amount of α-adrenergic (α-Ad) receptors or the high K +-stimulated contraction. The decrease in the number of muscarinic receptors was prevented by removing Ca 2+ from the culture medium with EGTA. The smooth muscle relaxant, papaverine, partially antagonized the effect of ACh on the number of receptors. When muscles were transferred to normal medium after treatment with ACh, the amount of m-ACh receptors increased very slowly, and this increase was prevented by cycloheximide (5 mM). Colchicine, an anti-microtubule alkaloid, suppressed both the ACh-induced decrease in the receptor number and increase in the receptor numbers after its decrease. The following suggestions are proposed from these results: 1. 1) The amount of m-ACh receptor determines the contractility of smooth muscle to ACh under physiological conditions. 2. 2) Receptor activation, and/or resultant muscle activity by ACh can regulate the amount of m-ACh receptor, but not α-Ad receptor. 3. 3) Microtubules seem to be involved in metabolic turnover of m-ACh receptor in smooth muscle.

A possible role of synaptic-membrane protein phosphorylation in the regulation of muscarinic acetylcholine receptors

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Long-term regulation of muscarinic acetylcholine receptors on cultured nerve cells

Incubation of mouse neuroblastoma cells (clone 1LE-115) with the muscarinic agonist, carbachol, resulted in a time-dependent desensitization to muscarinic receptor-mediated cyclic GMP formation and a decrease in the number and affinity of muscarinic receptors as measured by the binding to intact cells of the muscarinic antagonist, [ 3H]quinuclidinyl benzilate (QNB). The decrease in responsiveness to cyclic GMP formation reached a maximum after a 15-minute exposure to 1 mM carbachol (short-term desensitization) whereas changes in [ 3H] QNB binding became apparent only after a one hour exposure and reached a maximum after about 12 hrs (long-term desensitization). Recovery of sensitivity after short-term desensitization was rapid, suggesting that this process may involve a conformational change in the muscarinic receptor. In contrast, recovery after long-term desensitization was prolonged and could be slowed by the inhibition of protein synthesis. These results indicate that different cellular mechanisms are involved in the short-term and long-term desensitization of muscarinic receptors.

Muscarinic acetylcholine receptor regulation by accelerated rate of receptor loss

Sustained activation of muscarinic acetylcholine receptors on neuron-like NG108-15 hybrid cells reduces the number of [ 3H]quinuclidinyl benzilate binding sites per cell as much as 88%. The response occurs at concentrations of agonists commensurate with those needed to occupy receptors and inhibit adenylate cyclase. Decreases in steady-state receptor levels persist as long as activator remains present. Withdrawal of activator results in a slow increase in receptor levels that is blocked by cycloheximide. Activation shortens receptor half-life by a factor of nearly 4, indicating that regulation occurs at the level of receptor breakdown.