Capacitative Ca2+ entry is involved in regulating soluble amyloid precursor protein (sAPPalpha) 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.     

Distinct sequence elements control the specificity of G protein activation by muscarinic acetylcholine receptor subtypes.

Relatively little is understood concerning the mechanisms by which subtypes of receptors, G proteins and effector enzymes interact to transduce specific signals. Through expression of normal, hybrid and deletion mutant receptors in Xenopus oocytes, we determined the G protein coupling characteristics of the functionally distinct m2 and m3 muscarinic acetylcholine receptor (mAChR) subtypes and identified the critical receptor sequences responsible for G protein specificity. Activation of a pertussis toxin insensitive G protein pathway, leading to a rapid and transient release of intracellular Ca2+ characteristic of the m3 receptor, could be specified by the transfer of as few as nine amino acids from the m3 to the m2 receptor. In a reciprocal manner, transfer of no more than 21 residues from the m2 to the m3 receptor was sufficient to specify activation of a pertussis toxin sensitive G protein coupled to a slow and oscillatory Ca2+ release pathway typical of the m2 subtype. Notably, these critical residues occur within the same region of the third cytoplasmic domain of functionally distinct mAChR subtypes.     

Affinity profiles of various muscarinic antagonists for cloned human muscarinic acetylcholine receptor (mAChR) subtypes and mAChRs in rat heart and submandibular gland.

A family of five subtypes of muscarinic acetylcholine receptors (mAChR) has been identified based on their molecular structures and second signal transduction pathways. In the present study, we examined the antagonist binding profiles of 9 muscarinic antagonists (atropine, 4-DAMP, pirenzepine, oxybutynin, tiquizium, timepidium, propiverine, darifenacin and zamifenacin) for human muscarinic acetylcholine receptor subtypes (m1, m2, m3, m4 and m5) produced by using a baculovirus infection system in Sf9 insect cells, and rat tissue membrane preparations (heart and submandibular gland). In a scopolamine methyl chloride [N-methyl-3H]- ([3H]NMS) binding assay, pirenzepine and timepidium displayed the highest affinities for the m1 and m2 subtypes, respectively, and both zamifenacin and darifenacin had the highest affinities for the m3 subtype, although the selectivities among the five subtypes were less than 10-fold. Propiverine showed a slightly higher affinity for the m5 subtype, whereas none of the drugs used in this study was uniquely selective for the m4 subtype. The binding affinities of muscarinic antagonists for rat heart and submandibular gland strong correlated with those for human cloned m2 and m3 subtypes, respectively. These data suggest that [3H]NMS binding studies using rat heart and submandibular gland might be useful methods which predict the affinities of test drugs for human muscarinic M2 and M3 receptor subtypes.     

Multiple topological domains mediate subtype-specific internalization of the M2 muscarinic acetylcholine receptor

Endocytosis of agonist-activated G protein-coupled receptors (GPCRs) is required for both resensitization and recycling to the cell surface as well as lysosomal degradation. Thus, this process is crucial for regulation of receptor signaling and cellular responsiveness. Although many GPCRs internalize into clathrin-coated vesicles in a dynamin-dependent manner, some receptors, including the M(2) muscarinic acetylcholine receptor (mAChR), can also exhibit dynamin-independent internalization. We have identified five amino acids, located in the sixth and seventh transmembrane domains and the third intracellular loop, that are essential for agonist-induced M(2) mAChR internalization via a dynamin-independent mechanism in JEG-3 choriocarcinoma cells. Substitution of these residues into the M(1) mAChR, which does not internalize in these cells, is sufficient for conversion to the internalization-competent M(2) mAChR phenotype, whereas removal of these residues from the M(2) mAChR blocks internalization. Cotransfection of a dominant-negative isoform of dynamin has no effect on M(2) mAChR internalization. An internalization-incompetent M(2) mutant that lacks a subset of the necessary residues can still internalize via a G protein-coupled receptor kinase-2 and beta-arrestin-dependent pathway. Furthermore, internalization is independent of the signal transduction pathway that is activated. These results identify a novel motif that specifies structural requirements for subtype-specific dynamin-independent internalization of a GPCR.     

MUSCARINIC ACETYLCHOLINE RECEPTOR SUBTYPE mRNAs IN THE HUMAN AND RAT VESTIBULAR PERIPHERY

The expression of the five muscarinic acetylcholine receptor (mAChR) subtypes (m1–m5) in the vestibular end-organs and in the primary afferent vestibular ganglia of the human and rat was studied using RT-PCR from the two tissue populations from both species. In the human, although all five mAChR subtypes were expressed in brain, only the m1, m2, and m5 mAChR subtypes were amplified from both the vestibular ganglia and the vestibular end-organs, while in the rat, all five mAChR subtypes were expressed. These data suggest that the efferent cholinergic axo-dendritic and axo-somatic synapses have a muscarinic component and that there are pharmacologic implications for patients with vestibular dysfunction.     

Charged amino acids required for signal transduction by the m3 muscarinic acetylcholine receptor.

The five muscarinic acetylcholine receptor (mAChR) subtypes, termed m1-m5, transduce agonist signals across the plasma membrane by activating guanine nucleotide binding (G) proteins. The large cytoplasmic domain joining the fifth and sixth transmembrane segments of mAChRs plays a critical role in controlling the specificity of G protein coupling. In this study, we determined which sequences within this domain are required for activation of signaling by the m3 mAChR. By measuring the ability of normal and mutant m3 mAChRs to couple to the G protein pathway leading to activation of phospholipase C and Ca(2+)-dependent chloride currents in RNA-injected Xenopus oocytes, we found that two clusters of charged residues near the fifth and sixth transmembrane segments were required for normal signaling; furthermore, the position of these sequences was critical for their function. Finally, analysis of deletion mutant m3 mAChRs confirmed the importance of these sequences; receptors containing as few as 22 out of 239 amino acids of the cytoplasmic domain were fully active in signaling if they included the critical charged residues. Sequence comparisons suggest that similar charged sequences may be required for signal transduction by many G protein-coupled receptors.     

Tissue distribution of mRNAs encoding muscarinic acetylcholine receptor subtypes

The tissue distribution of the mRNAs encoding muscarinic acetylcholine receptors (mAChRs) I, II, III and IV has been investigated by blot hybridization analysis with specific probes. This study indicates that exocrine glands contain both mAChR I and III mRNAs, whereas smooth muscles contain both mAChR II and III mRNAs. All four mAChR mRNAs are present in cerebrum, whereas only mAChR II mRNA is found in heart.     

Calcium mobilization by muscarinic receptors in human astrocytoma cells: measurements with quin 2

Activation of muscarinic cholinergic receptors on 1321N1 human astrocytoma cells leads to Ca2+ mobilization as measured by quin 2 fluorescence. Acetylcholine and methacholine were full and potent agonists, while carbachol and muscarine, were fully efficacious but 6- and 10-fold less potent than acetylcholine. The carbachol-induced Ca2+ response was also observed in absence of extracellular Ca2+ and was blocked by muscarinic receptor antagonists but not by organic Ca2+ channel blockers, tetrodotoxin (TTX), tetraethylammonium (TEA) or metal cations, suggesting that Ca2+ is mobilized from intracellular storage sites rather than through plasma membrane ion channels. Muscarinic receptor-mediated Ca2+ release was also detected in kidney epithelial cells but not in rat fibroblasts, glial cells or differentiated neuroblastoma x glioma hybrid cells.     

Muscarinic toxin-like proteins from cobra venom.

Three new polypeptides were isolated from the venom of the Thailand cobra Naja kaouthia and their amino-acid sequences determined. They consist of 65-amino-acid residues and have four disulfide bridges. A comparison of the amino-acid sequences of the new polypeptides with those of snake toxins shows that two of them (MTLP-1 and MTLP-2) share a high degree of similarity (55-74% sequence identity) with muscarinic toxins from the mamba. The third polypeptide (MTLP-3) is similar to muscarinic toxins with respect to the position of cysteine residues and the size of the disulfide-confined loops, but shows less similarity to these toxins (30-34% sequence identity). It is almost identical with a neurotoxin-like protein from Bungarus multicinctus (TrEMBL accession number Q9W727), the sequence of which has been deduced from cloned cDNA only. The binding affinities of the isolated muscarinic toxin-like proteins towards the different muscarinic acetylcholine receptor (mAChR) subtypes (m1-m5) was determined in competition experiments with N-[3H]methylscopolamine using membrane preparations from CHO-K1 cells, which express these receptors. We found that MTLP-1 competed weakly with radioactive ligand for binding to all mAChR subtypes. The most pronounced effect was observed for the m3 subtype; here an IC50 value of about 3 microM was determined. MTLP-2 had no effect on ligand binding to any of the mAChR subtypes at concentrations up to 1 microM. MTLP-1 showed no inhibitory effect on alpha-cobratoxin binding to the nicotinic acetylcholine receptor from Torpedo californica at concentrations up to 20 microM.     

Acetylcholine induces mesenchymal stem cell migration via Ca2+ /PKC/ERK1/2 signal pathway

Acetylcholine (ACh) plays an important role in neural and non-neural function, but its role in mesenchymal stem cell (MSC) migration remains to be determined. In the present study, we have found that ACh induces MSC migration via muscarinic acetylcholine receptors (mAChRs). Among several mAChRs, MSCs express mAChR subtype 1 (m1AChR). ACh induces MSC migration via interaction with mAChR1. MEK1/2 inhibitor PD98059 blocks ERK1/2 phosphorylation while partially inhibiting the ACh-induced MSC migration. InsP3Rs inhibitor 2-APB that inhibits MAPK/ERK phosphorylation completely blocks ACh-mediated MSC migration. Interestingly, intracellular Ca(2+) ATPase-specific inhibitor thapsigargin also completely blocks ACh-induced MSC migration through the depletion of intracellular Ca(2+) storage. PKCα or PKCβ inhibitor or their siRNAs only partially inhibit ACh-induced MSC migration, but PKC-ζ siRNA completely inhibits ACh-induced MSC migration via blocking ERK1/2 phosphorylation. These results indicate that ACh induces MSC migration via Ca(2+), PKC, and ERK1/2 signal pathways.     

Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca2+]i) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of Galphaq/11-coupled M1, M3 and M5 receptors and intracellular calcium stores, whereas Galphai/o-protein coupled M2 receptor activity mediated neuronal differentiation.     

The Cysteine Residue in the Carboxyl-Terminal Domain of the m2 Muscarinic Acetylcholine Receptor Is Not Required for Receptor-Mediated Inhibition of Adenylate Cyclase

Muscarinic acetylcholine receptors (mAChRs) share with many other receptors of the guanine nucleotide-binding protein-coupled receptor family a highly conserved cysteine residue in the putative cytoplasmic carboxyl-terminal region of the protein. Because elimination of this cysteine in the beta 2-adrenergic receptor has been reported to decrease functional responsiveness, we determined if this cysteine residue is essential for mAChR-effector coupling by replacing Cys457 of the m2 mAChR with glycine and expressing wild-type and mutant receptor in Chinese hamster ovary (CHO) cells. The mutant and wild-type receptors exhibited similar affinities for binding of muscarinic ligands. In addition, the mutation did not affect cell surface localization or receptor-mediated inhibition of adenylate cyclase. These results indicate that the cysteine residue in the carboxyl-terminal domain of the m2 mAChR is not required for ligand binding or mAChR-mediated inhibition of adenylate cyclase in CHO cells.     

Embryonic chick heart expresses multiple muscarinic acetylcholine receptor subtypes. Isolation and characterization of a gene encoding a novel m2 muscarinic acetylcholine receptor with high affinity for pirenzepine.

Muscarinic acetylcholine receptors (mAChR) are G protein-coupled receptors which are highly conserved across mammalian species. Chick cardiac mAChR, however, have been shown to be pharmacologically, immunologically, and biochemically distinct from m2 mAChR expressed in mammalian heart. We previously reported the isolation and characterization of a novel chicken mAChR, cm4, which is expressed in chick heart and brain. We report here the isolation of an additional chicken mAChR gene whose deduced amino acid sequence is most homologous to the mammalian m2 receptor. Northern blot analysis demonstrated that this chicken m2 gene is also expressed in chick heart and brain. When stably transfected into Chinese hamster ovary (CHO) cells and Y1 adrenal carcinoma cells, the chicken m2 gene expresses a receptor protein which exhibits high affinity binding for the muscarinic antagonist quinuclidinyl benzilate and atropine, as well as the M1-selective antagonist pirenzepine and the M2-selective antagonist AF-DX 116. Therefore, when expressed in two heterologous cell lines, the chick m2 receptor has pharmacological properties that are similar to the chick m4 receptor as well as those reported for endogenous mAChR in chick cardiac cells. Consistent with the properties of the chick m4, as well as mammalian m2 and m4 receptors, the chick m2 receptor was able to functionally couple to both the inhibition of adenylate cyclase and the stimulation of phosphoinositide metabolism when expressed in CHO cells, but only the inhibition of adenylate cyclase when expressed in Y1 cells. We conclude from this study that the embryonic chick heart expresses multiple subtypes of mAChR which are highly conserved with their mammalian counterparts. Furthermore, the high degree of conservation between the mammalian m2 and the chick m2 muscarinic receptors suggests that the pharmacological differences that exist between these receptors are due to a relatively small number of specific amino acid changes rather than larger changes in receptor sequence or structure.     

Regulation of signal transduction at M2 muscarinic receptor

Muscarinic acetylcholine receptors mediate transmission of an extracellular signal represented by released acetylcholine to neuronal or effector cells. There are five subtypes of closely homologous muscarinic receptors which are coupled by means of heterotrimeric G-proteins to a variety of signaling pathways resulting in a multitude of target cell effects. Endogenous agonist acetylcholine does not discriminate among individual subtypes and due to the close homology of the orthosteric binding site the same holds true for most of exogenous agonists. In addition to the classical binding site muscarinic receptors have one or more allosteric binding sites at extracellular domains. Binding of allosteric modulators induces conformational changes in the receptor that result in subtype-specific changes in orthosteric binding site affinity for both muscarinic agonists and antagonists. This overview summarizes our recent experimental effort in investigating certain aspects of M2 muscarinic receptor functioning concerning i) the molecular determinants that contribute to the binding of allosteric modulators, ii) G-protein coupling specificity and subsequent cellular responses and iii) possible functional assays that exploit the unique properties of allosteric modulators for characterization of muscarinic receptor subtypes in intact tissue. A detailed knowledge of allosteric properties of muscarinic receptors is required to permit drug design that will modulate signal transmission strength of specific muscarinic receptor subtypes. Furthermore, allosteric modulation of signal transmission strength is determined by cooperativity rather than concentration of allosteric modulator and thus reduces the danger of overdose.     

Muscarinic receptor enhancement of nicotine-induced catecholamine secretion may be mediated by phosphoinositide metabolism in bovine adrenal chromaffin cells

Bovine adrenal chromaffin cells possess both nicotinic and muscarinic cholinergic receptors, but only nicotinic receptors have heretofore appeared to mediate Ca2+-dependent exocytosis. We have now found that muscarinic receptor stimulation in bovine adrenal chromaffin cells leads to enhanced inositol phospholipid metabolism as evidenced by the rapid (less than 1 min) formation of inositol trisphosphate (IP3) and inositol bisphosphate (IP2). Muscarinic receptor-mediated accumulation of IP3 and IP2 continues beyond 1 min in the presence of LiCl and is accompanied by large increases in inositol monophosphate. Muscarinic receptor stimulation was also found to enhance nicotine-induced catecholamine secretion by 1.7-fold if muscarine was added 30 s before nicotine addition. Moreover, since the muscarinic antagonist atropine reduces acetylcholine-induced secretion, we conclude that muscarinic receptor stimulation somehow primes these cells for nicotinic receptor-mediated secretion, perhaps by causing small nonstimulatory increases in cytosolic free Ca2+ mediated by IP3. Furthermore, we show that small depolarizations of these cells with 10 mM K+, which themselves do not affect basal secretion, also enhance nicotine-induced secretion. Thus, small increases in cytosolic free Ca2+ produced either by physiologic muscarinic receptor stimulation or by small experimental depolarizations with K+ may prime the chromaffin cells for nicotinic receptor-mediated secretion.     

Activation of M3 muscarinic acetylcholine receptors inhibits voltage-dependent calcium influx in small cell lung carcinoma

Small cell carcinoma of the lung (SCC) expresses several characteristics of neuronal cells, including voltage-gated Ca2+ channels (VGCC), and also expresses muscarinic acetylcholine receptors (mAChR). In testing the possibility that VGCC may be functionally coupled to mAChR in SCC cell lines, we found that depolarization-dependent Ca2+ influx was inhibited by carbachol (IC50 = 0.78 microM) and oxotremorine (IC50 = 0.69 microM). Equilibrium dissociation constants for several mAChR antagonists indicated that a mAChR of M3 subtype was involved. Exposure of SCC to carbachol induced the hydrolysis of phosphoinositides and increased the cytosolic free Ca2+ concentration ([Ca2+]i). The carbachol-mediated inhibition of depolarization-dependent Ca2+ influx did not directly correlate with increased [Ca2+]i but did correlate with inositol poly-phosphate generation. The protein kinase C activators phorbol 12-myristate 13-acetate or 1-oleoyl-2-acetyl-sn-glycerol neither mimicked nor amplified the inhibitory effect of carbachol on Ca2+ influx. However, phorbol 12-myristate 13-acetate suppressed the carbachol-induced inositol polyphosphate generation and inhibition of depolarization-dependent Ca2+ influx. The inactive compound 4 alpha-phorbol had no effect. These data suggest that the inhibition of VGCC caused by carbachol is not due to protein kinase C activation, but rather is due to events mediated by inositol polyphosphates. This is the first documentation of a role for phosphoinositide hydrolysis in the functional coupling of mAChR and VGCC. The expression of M3 mAChR functionally coupled to VGCC could have therapeutic implications for SCC, in light of recent demonstrations that cell proliferation can be influenced by activation of neurotransmitter receptors.     

Muscarinic acetylcholine receptors stimulate Ca2+ influx in PC12D cells predominantly via activation of Ca2+ store-operated channels

Activation of muscarinic acetylcholine receptors (mAChRs) causes the rapid release of Ca2+ from intracellular stores and a sustained influx of external Ca2+ in PC12D cells, a subline of the widely studied cell line PC12. Release of Ca2+ from intracellular stores and a sustained influx of Ca2+ are also observed following exposure to thapsigargin, a sesquiterpene lactone that depletes intracellular Ca2+ pools by irreversibly inhibiting the Ca2+ pump of the endoplasmic reticulum. In this study, we show that carbachol and thapsigargin empty the same intracellular Ca2+ stores, and that these stores are a subset of intracellular stores depleted by the Ca2+ ionophore ionomycin. Intracellular Ca2+ stores remain depleted during continuous stimulation of mAChR with carbachol in medium containing 2 mM extracellular Ca2+, but rapidly refill following inhibition of mAChRs with atropine. Addition of atropine to carbachol-stimulated cells causes intracellular Ca2+ levels to return to baseline levels in two steps: a rapid decrease that correlates with the reuptake of Ca2+ into internal stores and a delayed decrease that correlates with the inhibition of a Mn2+-permeable Ca2+ channel. Several lines of evidence suggest that carbachol and thapsigargin stimulate Ca2+ influx by a common mechanism: (i) pretreatment with thapsigargin occludes atropine-mediated inhibition of Ca2+ influx, (ii) carbachol and thapsigargin applied individually or together are equally efficient at stimulating the influx of Mn2+, and (iii) identical rates of Ca2+ influx are observed when Ca2+ is added to cells pretreated with carbachol, thapsigargin, or both agents in the absence of extracellular Ca2+. Taken together, these data suggest that the sustained influx of extracellular Ca2+ observed following activation of mAChRs in PC12D cells is mediated primarily by activation of a Mn2+-permeable, Ca2+ store-operated Ca2+ channel.