Stimulation Of mAChRs Research Articles

Carbofuran pesticide toxicity to the eye

Pesticide exposure to eyes is a major source of ocular morbidities in adults and children all over the world. Carbofuran (CF), N-methyl carbamate, pesticide is most widely used as an insecticide, nematicide, and acaricide in agriculture, forestry, and gardening. Contact or ingestion of carbofuran causes high morbidity and mortality in humans and pets. Pesticides are absorbed in the eye faster than other organs of the body and damage ocular tissues very quickly. Carbofuran exposure to eye causes blurred vision, pain, loss of coordination, anti-cholinesterase activities, weakness, sweating, nausea and vomiting, abdominal pain, endocrine, reproductive, and cytotoxic effects in humans depending on amount and duration of exposure. Pesticide exposure to eye injures cornea, conjunctiva, lens, retina, and optic nerve and leads to abnormal ocular movement and vision impairment. Additionally, anticholinesterase pesticides like carbofuran are known to cause salivation, lacrimation, urination, and defecation (SLUD). Carbofuran and its two major metabolites (3-hydroxycarbofuran and 3-ketocarbofuran) are reversible inhibitors of acetylcholinesterase (AChE) which regulates acetylcholine (ACh), a neurohumoral chemical that plays an important role in corneal wound healing. The corneal epithelium contains high levels of ACh whose accumulation by AChE inhibition after CF exposure overstimulates muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs). Hyper stimulation of mAChRs in the eye causes miosis (excessive constriction of the pupil), dacryorrhea (excessive flow of tears), or chromodacryorrhea (red tears). Recent studies reported alteration of autophagy mechanism in human cornea in vitro and ex vivo post carbofuran exposure. This review describes carbofuran toxicity to the eye with special emphasis on corneal morbidities and blindness.

Protection from interferon-β-induced neuronal apoptosis through stimulation of muscarinic acetylcholine receptors coupled to ERK1/2 activation.

Although clinically useful for their immunomodulatory, antiproliferative and antiviral properties, type I interferons (IFNs) are involved in the pathogenesis of several neurodegenerative/neuroinflammatory diseases. In the present study, we investigated the ability of cholinergic stimulation to protect from IFN-β-induced neuronal apoptosis. The effects of the ACh receptor agonist carbachol (CCh) on IFN-β-induced apoptosis of human SH-SY5Y neuroblastoma cells were examined by using western blots, immunofluorescence and cytofluorimetry. The involvement of muscarinic acetylcholine receptors (mAChRs) was assessed by using selective antagonists and siRNA transfection. Pharmacological inhibitors and overexpression of ERK2 and an ERK2 constitutively active form (ERK2-CA) were employed to study ERK1/2 signalling. The effects of oxotremorine-M (Oxo-M) on IFN-β-induced apoptosis of mouse hippocampal neurons were examined by measuring cleaved caspase 3 expression. In SH-SY5Y cells, CCh inhibited IFN-β-induced mitochondrial cytochrome c release, activation of caspases 9, 7 and 3, PARP cleavage and DNA fragmentation. The anti-apoptotic effect of CCh was mediated by M3 receptors, blocked by Gq/11 antagonist YM254890 and PKC inhibitor Go 6983, impaired by inhibition of ERK1/2 pathway, potentiated by overexpression of ERK2 and mimicked by ERK2-CA. Blockade of JNK activation enhanced the CCh anti-apoptotic response. IFN-β inhibited JNK activation and up-regulated CCh-induced ERK1/2 signalling. In hippocampal neurons, Oxo-M reduced IFN-β-induced apoptosis; this effect was antagonized by blockade of M1 /M3 receptors and ERK1/2. Stimulation of mAChRs counteracted IFN-β-induced neuronal apoptosis through the activation of ERK1/2 signalling. The data indicate that activation of ERK1/2-coupled mAChRs may be an effective strategy for preventing IFNs neurotoxicity.

Muscarinic Receptors as Targets for Metronomic Therapy in Breast Cancer.

It is actually known that acetylcholine works as a signaling molecule in non-neuronal cells and tissues, in addition to its neuronal function as neurotransmitter. It can act on two types of receptors nicotinic and muscarinic receptors (mAChRs). The latter belong to the G protein coupled receptor family and there are five subtypes genetically cloned. Their activation triggers classical and non-classical intracellular signals that could be linked to the proliferation of normal and/or transformed cells. The M3 subtype was identified in different types of tumors and its stimulation with agonists triggers cell proliferation, migration, invasion and metastasis. Our laboratory has extensively investigated the expression and function of mAChRs in breast tumors from animal and human origins. We found a profuse expression of mAChRs in breast tumors, but opposite to this, an absence of these receptors in normal breast cells and tissues. The stimulation of mAChRs with the cholinergic agonist carbachol for 20 h increased tumor cell death. Moreover, the combination of subthreshold concentrations of the agonist with paclitaxel potentiates cell death. The usage of low dose chemotherapy with short drug free intervals was named metronomic therapy and it has emerged as a novel regimen for cancer treatment with very low incidence of side effects. Our work and that of others indicate that mAChRs that are over-expressed in different types of tumor cells could be a useful target for metronomic therapy in cancer treatment.

Developmental nicotine exposure alters cholinergic control of respiratory frequency in neonatal rats

Prenatal nicotine exposure with continued exposure through breast milk over the first week of life (developmental nicotine exposure, DNE) alters the development of brainstem circuits that control breathing. Here, we test the hypothesis that DNE alters the respiratory motor response to endogenous and exogenous acetylcholine (ACh) in neonatal rats. We used the brainstem-spinal cord preparation in the split-bath configuration, and applied drugs to the brainstem compartment while measuring the burst frequency and amplitude of the fourth cervical ventral nerve roots (C4VR), which contain the axons of phrenic motoneurons. We applied ACh alone; the nicotinic acetylcholine receptor (nAChR) antagonist curare, either alone or in the presence of ACh; and the muscarinic acetylcholine receptor (mAChR) antagonist atropine, either alone or in the presence of ACh. The main findings include: (1) atropine reduced frequency similarly in controls and DNE animals, while curare caused modest slowing in controls but no consistent change in DNE animals; (2) DNE greatly attenuated the increase in C4VR frequency mediated by exogenous ACh; (3) stimulation of nAChRs with ACh in the presence of atropine increased frequency markedly in controls, but not DNE animals; (4) stimulation of mAChRs with ACh in the presence of curare caused a modest increase in frequency, with no treatment group differences. DNE blunts the response of the respiratory central pattern generator to exogenous ACh, consistent with reduced availability of functionally competent nAChRs; DNE did not alter the muscarinic control of respiratory motor output. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1138-1149, 2016.

The acetylcholine signaling network of corneal epithelium and its role in regulation of random and directional migration of corneal epithelial cells.

Because cholinergic drugs are used in ophthalmology and cholinergic stimulation has been shown to facilitate epithelialization of mucocutaneous wounds, we performed a systematic analysis of components of the cholinergic network of human and murine corneal epithelial cells (CECs) and determined the role of autocrine and paracrine acetylcholine (ACh) in regulation of CEC motility. We investigated the expression of ACh receptors at the mRNA and protein levels in human immortalized CECs, localization of cholinergic molecules in normal and wounded murine cornea, and the effects of cholinergic drugs on CEC directional and random migration in vitro, intercellular adhesion, and expression of integrin αV and E-cadherin. We demonstrated that corneal epithelium expresses the ACh-synthesizing enzyme choline acetyltransferase, the ACh-degrading enzyme acetylcholinesterase, two muscarinic ACh receptors (mAChRs), M3 and M4, and several nicotinic ACh receptors (nAChRs), including both α7- and α9-made homomeric nAChRs and predominantly the α3β2±α5 subtype of heteromeric nAChRs. Wounding affected the expression patterns of cholinergic molecules in the murine corneal epithelium. Constant stimulation of CECs through both muscarinic and nicotinic signaling pathways was essential for CEC survival and both directional and random migration in vitro. Both α7 and non-α7 nAChRs elicited chemotaxis, with the α7 signaling exhibiting a stronger chemotactic effect. Cholinergic stimulation of CECs upregulated expression of the integrin and cadherin molecules involved in epithelialization. We found synergy between the proepithelialization signals elicited by different ACh receptors expressed in CECs. Simultaneous stimulation of mAChRs and nAChRs by ACh may be required to synchronize and balance ionic and metabolic events in a single cell. Localization of these cholinergic enzymes and receptors in murine cornea indicated that the concentration of endogenous ACh and the mode of its signaling differ among corneal epithelial layers. Elucidation of the signaling events elicited upon agonist binding to corneal mAChRs and nAChRs will be crucial for understanding the mechanisms of ACh signaling in CECs, which has salient clinical implications.

Hippocampal Gαq/11 but not Gαo-coupled receptors are altered in aging

Normal aging may limit the signaling efficacy of certain GPCRs by disturbing the function of specific Gα-subunits and leading to deficient modulation of intracellular functions that subserve synaptic plasticity, learning and memory. Evidence suggests that Gαq/11 is more sensitive to the effects of aging relative to other Gα-subunits, including Gαo. To test this hypothesis, the functionality of Gαq/11 and Gαo were compared in the hippocampus of young (6 months) and aged (24 months) F344 × BNF1 hybrid rats assessed for spatial learning ability. Basal GTPγS-binding to Gαq/11 was significantly elevated in aged rats relative to young and but not reliably associated with spatial learning. mAChR stimulation of Gαq/11 with oxotremorine-M produced equivocal GTPγS-binding between age groups although values tended to be lower in the aged hippocampus and were inversely related to basal activity. Downstream Gαq/11 function was measured in hippocampal subregion CA1 by determining changes in [Ca2+]i after mAChR and mGluR (DHPG) stimulation. mAChR-stimulated peak change in [Ca2+]i was lower in aged CA1 relative to young while mGluR-mediated integrated [Ca2+]i responses tended to be larger in aged. GPCR modulation of [Ca2+]i was observed to depend on intracellular stores to a greater degree in aged than young. In contrast, measures of Gαo-mediated GTPγS-binding were stable across age, including basal, mAChR-, GABABR (baclofen)-stimulated levels. Overall, the data indicate that aging selectively modulates the activity of Gαq/11 within the hippocampus leading to deficient modulation of [Ca2+]i following stimulation of mAChRs but these changes are not related to spatial learning.

Diminished antigen-specific IgG 1 and interleukin-6 production and acetylcholinesterase expression in combined M 1 and M 5 muscarinic acetylcholine receptor knockout mice

Immunological activation of T cells enhances synthesis of acetylcholine (ACh) and transcription of choline acetyltransferase (ChAT), M 5 muscarinic ACh receptor (mAChR) and acetylcholinesterase (AChE). Stimulation of mAChRs on T and B cells causes oscillating Ca 2+-signaling and up-regulation of c-fos expression; moreover, M 1 mAChRs play a crucial role in the differentiation of CD8 + T cells into cytolytic T lymphocytes. Collectively, these findings suggest that immune cell function is regulated by its own cholinergic system. Bearing that in mind, we tested whether immune function can be regulated via mAChR-mediated pathways by immunizing combined M 1 and M 5 mAChR knockout (M 1/M 5 KO) and wild-type (WT) C57BL/6JJcl mice with ovalbumin (OVA) and measuring serum IgG 1 and IgM 1 wk later. We found that serum levels of total and anti-OVA-specific IgG 1 were significantly lower in M 1/M 5 KO than WT mice, though there was no difference in serum levels of total and anti-OVA-specific IgM between the two genotypes. Secretion of interleukin (IL)-6 from activated spleen cells was significantly reduced in M 1/M 5 KO mice, whereas there was no significant change in gamma interferon secretion. Expression of AChE mRNA was significantly reduced in activated spleen cells from M 1/M 5 KO mice. These results suggest that M 1 and/or M 5 mAChRs are involved in regulating cytokine ( e.g., IL-6) production, leading to modulation of antibody class switching from IgM to IgG 1, but are not involved in the initial generation of the antibody response. They also support the notion that a non-neuronal cholinergic system is involved in regulating immune cell function.

Agonist mediated internalization of M<sub>2</sub> mAChR is β-arrestin-dependent

BackgroundMuscarinic acetylcholine receptors (mAChRs) undergo agonist-promoted internalization, but evidence suggesting that the mechanism of internalization is β-arrestin dependent has been contradictory and unclear. Previous studies using heterologous over-expression of wild type or dominant-negative forms of β-arrestins have reported that agonist-promoted internalization of M2 mAChRs is a β-arrestin- and clathrin-independent phenomenon. In order to circumvent the complications associated with the presence of endogenous β-arrestin that may have existed in these earlier studies, we examined agonist-promoted internalization of the M2 mAChR in mouse embryonic fibroblasts (MEFs) derived from β-arrestin knockout mice that lack expression of either one or both isoforms of β-arrestin (β-arrestin 1 and 2).ResultsIn wild type MEF cells transiently expressing M2 mAChRs, 40% of surface M2 mAChRs underwent internalization and sorted into intracellular compartments following agonist stimulation. In contrast, M2 mAChRs failed to undergo internalization and sorting into intracellular compartments in MEF β-arrestin double knockout cells following agonist stimulation. In double knockout cells, expression of either β-arrestin 1 or 2 isoforms resulted in rescue of agonist-promoted internalization. Stimulation of M2 mAChRs led to a stable co-localization with GFP-tagged β-arrestin within endocytic structures in multiple cell lines; the compartment to which β-arrestin localized was determined to be the early endosome. Agonist-promoted internalization of M2 mAChRs was moderately rescued in MEF β-arrestin 1 and 2 double knockout cells expressing exogenous arrestin mutants that were selectively defective in interactions with clathrin (β-arrestin 2 ΔLIELD), AP-2 (β-arrestin 2-F391A), or both clathrin/AP-2. Expression of a truncated carboxy-terminal region of β-arrestin 1 (319–418) completely abrogated agonist-promoted internalization of M2 mAChRs in wild type MEF cells.ConclusionIn summary, this study demonstrates that agonist-promoted internalization of M2 mAChRs is β-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with β-arrestin in early endosomal vesicles.

Muscarinic Acetylcholine Receptors Activate TRPC6 Channels in PC12D Cells via Ca2+ Store–Independent Mechanisms

In this paper we report that stimulation of mAChRs in PC12D cells activates Ca2+ channels that are regulated independently of intracellular Ca2+ stores. In nominally Ca2+-free medium, exposure of PC12D cells to carbachol stimulates a robust influx of Ba2+, a Ca2+ substitute. This influx is blocked by atropine, but not by inhibitors of the nicotinic acetylcholine receptor or L-, N-, or T-type voltage-regulated Ca2+ channels. By contrast, depletion of intracellular Ca2+ stores with thapsigargin only weakly stimulates Ba2+ influx. Unlike store-operated Ca2+ channels (SOCCs), which close only after intracellular Ca2+ stores refill, channels mediating carbachol-stimulated Ba2+ influx rapidly close following the inactivation of mAChRs with atropine. Ba2+ influx is inhibited by extracellular Ca2+, by the Ca2+ channel blocker SKF-96365, and by activation of protein kinase C (PKC). Exogenous expression of antisense RNA encoding the rat canonical-transient receptor potential Ca2+ channel subtype 6 (TRPC6) or the N-terminal domain of TRPC6 blocks carbachol-stimulated Ba2+ influx in PC12D cells. Expression of TRPC6 antisense RNA or the TRPC6 N-terminal domain also blocks Ba2+ influx stimulated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a diacylglycerol analog previously shown to activate exogenously expressed TRPC6 channels. These data show that mAChRs in PC12D cells activate endogenous Ca2+ channels that are regulated independently of Ca2+ stores and require the expression of TRPC6.

Dynamic regulation of glycinergic input to spinal dorsal horn neurones by muscarinic receptor subtypes in rats

Activation of spinal muscarinic acetylcholine receptors (mAChRs) inhibits nociception. However, the cellular mechanisms of this action are not fully known. In this study, we determined the role of mAChR subtypes in regulation of synaptic glycine release in the spinal cord. Whole-cell voltage-clamp recordings were performed on lamina II neurones in the rat spinal cord slices. The mAChR agonist oxotremorine-M significantly increased the frequency of glycinergic sIPSCs but not mIPSCs. Surprisingly, the effect of oxotremorine-M on sIPSCs was largely attenuated at a higher concentration. On the other hand, 1-10 microm oxotremorine-M dose-dependently increased the frequency of sIPSCs in rats pretreated with intrathecal pertussis toxin. Furthermore, oxotremorine-M also dose-dependently increased the frequency of sIPSCs in the presence of himbacine (an M2/M4 mAChR antagonist) or AF-DX116 (an M2 mAChR antagonist). The M3 mAChR antagonist 4-DAMP abolished the stimulatory effect of oxotremorine-M on sIPSCs. Interestingly, the GABA(B) receptor antagonist CGP55845 potentiated the stimulatory effect of oxotremorine-M on sIPSCs. In the presence of CGP55845, both himbacine and AF-DX116 similarly reduced the potentiating effect of oxotremorine-M on sIPSCs. Collectively, these data suggest that the M3 subtype is present on the somatodendritic site of glycinergic neurones and is mainly responsible for muscarinic potentiation of glycinergic input to spinal dorsal horn neurones. Concurrent stimulation of mAChRs on adjacent GABAergic interneurones attenuates synaptic glycine release through presynaptic GABA(B) receptors on glycinergic interneurones. This study illustrates a complex dynamic interaction between GABAergic and glycinergic synapses in the spinal cord dorsal horn.

Activation of M1 Muscarinic Acetylcholine Receptors Stimulates the Formation of a Multiprotein Complex Centered on TRPC6 Channels

In this study we showed that stimulation of M1 muscarinic acetylcholine receptors (mAChRs) activates endogenous transient receptor potential-canonical, subtype 6 (TRPC6), channels in neuronal PC12D cells. Activation of TRPC6 channels is correlated with the formation of a multiprotein complex containing M1 mAChRs, TRPC6 channels, and protein kinase C (PKC). Formation of the M1 mAChR-TRPC6-PKC complex is transient, with highest levels reached approximately 2 min after stimulation of M1 mAChRs. PKC in the complex phosphorylates TRPC6 on a conserved serine residue in the carboxyl-terminal domain (Ser768 in the TRPC6A isoform and Ser714 in the TRPC6B isoform). The immunophilin FKBP12, the phosphatase calcineurin, and Ca2+-binding protein calmodulin are also recruited to the M1 mAChR-TRPC6-PKC complex following activation of M1 mAChRs and remain stably associated with the TRPC6 channels after M1 mAChRs and PKC have disassociated. Binding of FKBP12, calcineurin, and calmodulin to TRPC6 channels is blocked by the following: 1) inhibition of PKC; 2) mutation of the PKC phosphorylation site (Ser(7168/714)) in the channels; or 3) pretreatment with FK506 or rapamycin, immunosuppressants that directly bind FKBP12. Inhibition of FKBP12 binding blocks the dephosphorylation of TRPC6 channels and the disassociation of M1 mAChRs, without affecting disassociation of PKC. The calcineurin inhibitor cyclosporin A also blocks the dephosphorylation of TRPC6 and prevents the disassociation of M1 mAChRs. Together, these results show that activated TRPC6 channels form the center of a dynamic multiprotein complex that includes PKC and calcineurin, which respectively phosphorylate and dephosphorylate the channels. Phosphorylation of the TRPC6 channels by PKC is required for the binding of FKBP12, which in turn is required for the binding of calcineurin and calmodulin. Subsequent dephosphorylation of the channels by calcineurin is required for the disassociation of M1 mAChRs.

Nicotinic antagonists in the treatment of nerve agent intoxication

Nicotinic antagonists in the treatment of nerve agent intoxication

Desensitization of α7 nicotinic receptors potentiated the inhibitory effect on M-current induced by stimulation of muscarinic receptors in rat superior cervical ganglion neurons

Whole-cell patch-clamp recording from rat superior cervical ganglion neurons in culture was used to investigate the modulatory effect of desensitized alpha7-nAChRs on mAChRs. An inward alpha-bungarotoxin and methyllycaconitine sensitive current was elicited by rapid application of choline, which consisted of a fast and a slow desensitizing component. The amplitude of choline-evoked currents recorded 0.5, 1, 2, and 3 min after the prolonged application of choline (10 mM, 30 s) decreased to 25.3 +/- 9.2%, 45.9 +/- 11.8%, 66.3 +/- 14.5%, and 73.9 +/- 13.3% of their baseline levels, respectively. The amplitudes of M-currents, recorded at the same time intervals after the similar prolonged stimulation with choline, were decreased to 52.7 +/- 17.4%, 63.9 +/- 4.2%, 70.9 +/- 2.8%, and 72.9 +/- 17.3% of initial values respectively by focal application of pilocarpine (1 mM, 5 s) onto the soma of neurons. By contrast, before the desensitization of alpha7-nAChRs, M-currents were only decreased to 79.8 +/- 13.7% of baseline levels by pilocarpine (1 mM, 5 s). Whereas the desensitization of alpha7-nAChRs had no direct effects on M-currents, and the facilitated effects on muscarinic agonists on the M-currents induced by desensitized alpha7-nAChRs, were removed in the presence of alpha-bungarotoxin and methyllycaconitine. These results indicated that desensitization of alpha7-nAChRs could potentiate the inhibitory effect on M-current by stimulation of mAChRs with their agonist.

An independent, non-neuronal cholinergic system in lymphocytes and its roles in regulation of immune function

Acetylcholine (ACh) is classically thought of as a neurotransmitter in mammalian species. However, lymphocytes express most of the cholinergic components found in the nervous system, including ACh, choline acetyltransferase (ChAT), high-affinity choline transporter, and acetylcholinesterase as well as both muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively). Activation of T cells via the T cell receptor/CD3 complex, contact of T cells with antigen presenting cells, or activation of the adenylyl cyclase pathway in T cells modulates cholinergic activity, as evidenced by up-regulation of ChAT and M(5) mAChR mRNA expression. Stimulation of mAChRs on T and B cells with ACh or another mAChR agonists elicits intracellular Ca(2+) signaling, up-regulation of c-fos expression, increased nitric oxide synthesis and interleukin-2-induced signal transduction via M(3) and M(5) mAChR-mediated pathways. Acute stimulation of nAChRs with ACh or nicotine causes rapid and transient Ca(2+) signaling in T and B cells, probably via alpha7 nAChRs subunit-mediated pathways. Chronic nicotine stimulation, by contrast, down-regulates nAChR expression and suppresses T cell activity. Abnormalities in lymphocytic cholinergic system have been seen in animal models of immune deficiency and immune acceleration. Collectively, these data provided a compelling picture in which immune function is, at least partly, under the control of an independent, non-neuronal cholinergic system in lymphocytes.

Novel insight into the mechanisms involved in the regulation of the m1 muscarinic receptor, iNOS and nNOS mRNA levels

In this paper we have determined the different signaling pathways involved in M 1 muscarinic acetylcholine receptor (mAChR)-dependent stimulation of m1 mAChRs, neural and inducible isoforms of nitric oxide synthase (nNOS and iNOS)-mRNA gene expression of rat frontal cortex. Carbachol-stimulation of M 1 mAChRs exerts an increase in m1 mAChR-mRNA, activation of phosphoinositide (PI) turnover, translocation of protein kinase C (PKC) and stimulation of NOS activity. Inhibitors of phospholipase C (PLC), calcium/calmodulin and NOS, but not guanylate cyclase, prevent the carbachol-dependent increase of m1 mAChR-mRNA levels. These inhibitors also attenuate the muscarinic receptor-dependent increase in nNOS and iNOS mRNA levels. These results suggest that carbachol-activation of M 1 mAChRs increases m1 mAChR, nNOS and iNOS mRNA levels associated with increased production of nitric oxide (NO). The mechanism appears to occur secondarily to stimulation of PI turnover via PLC activation. This in turn, triggers a cascade reaction involving calcium/calmodulin and PKC, leading to activation of NOS. On the basis of our results, the activation of M 1 mAChRs appears to induce nNOS and iNOS expression and, reciprocally, the activator of NOS up-regulates m1 mAChR gene expression. These results may contribute to a better understanding of the effects and side effects of cholinomimetic treatment in patients with neurodegenerative diseases.

Stimulation of M3 Muscarinic Receptors Induces Phosphorylation of the Cdc42 Effector Activated Cdc42Hs-associated Kinase-1 via a Fyn Tyrosine Kinase Signaling Pathway

The tyrosine kinase, activated Cdc42Hs-associated kinase-1 (ACK-1), is a specific effector of the Rho family GTPase Cdc42. GTP-bound Cdc42 has been shown to facilitate neurite outgrowth elicited by activation of muscarinic cholinergic receptors (mAChRs). Because tyrosine kinase activity is a requirement for neuritogenesis in several cell systems, we investigated whether endogenous mAChRs (principally of the M3 subtype) expressed in human SH-SY5Y neuroblastoma cells would signal to ACK-1. Incubation of cells with the cholinergic agonist oxotremorine-M (Oxo-M) induced an approximately 6-fold increase in the tyrosine phosphorylation of ACK-1 which was inhibited by atropine. ACK-1 phosphorylation was blocked by Clostridium difficile toxin B, an inhibitor of Rho family GTPases. In contrast, disruption of the actin cytoskeleton with cytochalasin D stimulated ACK-1 phosphorylation, and moreover, addition of Oxo-M to cells preincubated with this agent elicited a further increase in phosphorylation, indicating that an intact cytoskeleton is not required for mAChR signaling to ACK-1. Although stimulation of M3 mAChRs induces both an increase in intracellular Ca2+ and activation of protein kinase C (PKC), neither of these second messenger pathways was required for receptor-stimulated ACK-1 phosphorylation. Instead, inhibition of PKC resulted in a 2-fold increase in Oxo-M-stimulated ACK-1 phosphorylation, whereas acute activation of PKC with phorbol ester decreased ACK-1 phosphorylation. The agonist-induced tyrosine phosphorylation of ACK-1 was blocked by inhibitors of Src family kinases, and ACK-1 was coprecipitated with Fyn (but not Src) in an agonist-dependent manner. Finally, scrape loading cells with glutathione S-transferase fusion proteins of either the Fyn-SH2 or Fyn-SH3 domain significantly attenuated mAChR-stimulated ACK-1 tyrosine phosphorylation. The data are the first to show phosphorylation of ACK-1 after stimulation of a receptor coupled to neurite outgrowth and indicate that a Rho family GTPase (i.e. Cdc42) and Fyn are essential upstream elements of this signaling pathway.

Muscarinic Acetylcholine Receptors Induce the Expression of the Immediate Early Growth Regulatory Gene CYR61

In brain, muscarinic acetylcholine receptors (mAChRs) modulate neuronal functions including long term potentiation and synaptic plasticity in neuronal circuits that are involved in learning and memory formation. To identify mAChR-inducible genes, we used a differential display approach and found that mAChRs rapidly induced transcription of the immediate early gene CYR61 in HEK 293 cells with a maximum expression after 1 h of receptor stimulation. CYR61 is a member of the emerging CCN gene family that includes CYR61/CEF10, CTGF/FISP-12, and NOV; these encode secretory growth regulatory proteins with distinct functions in cell proliferation, migration, adhesion, and survival. We found that CYR61, CTGF, and NOV were expressed throughout the human central nervous system. Stimulation of mAChRs induced CYR61 expression in primary neurons and rat brain where CYR61 mRNA was detected in cortical layers V and VI and in thalamic nuclei. In contrast, CTGF and NOV expression was not altered by mAChRs neither in neuronal tissue culture nor rat brain. Receptor subtype analyses demonstrated that m1 and m3 mAChR subtypes strongly induced CYR61 expression, whereas m2 and m4 mAChRs had only subtle effects. Increased CYR61 expression was coupled to mAChRs by both protein kinase C and elevations of intracellular Ca(2+). Our results establish that CYR61 expression in mammalian brain is under the control of cholinergic neurotransmission; it may thus be involved in cholinergic regulation of synaptic plasticity.

M1 muscarinic acetylcholine receptors activate zif268 gene expression via small G-protein Rho-dependent and lambda-independent pathways in PC12D cells.

We have previously shown that stimulation of M1 muscarinic acetylcholine receptors (mAChRs) in neuronal PC12D cells rapidly induces the immediate-early gene zif 268 [Ebihara, T. & Saffen, D. (1997) J. Neurochem. 68, 1001-1010]. Here we show that stimulation of M1 mAChRs in these cells activates four distal serum response elements (SREs) in the zif 268 promoter, and that this activation is strongly inhibited by Clostridium botulinum C3 exoenzyme (C3), which specifically inactivates the small G-protein Rho. Even with high doses of C3, however, a portion of the activation remains intact, indicating that stimulation of M1 mAChRs activates zif 268 SREs via Rho-dependent and Rho-independent pathways. Moreover, the Rho-independent activation of zif 268 SREs is inhibited by the dominant-negative form of the small G-protein Ras, suggesting that Rho-independent activation of zif 268 SREs is mediated by Ras. To determine if muscarinic agonists activate RhoA, we also measured the translocation of RhoA from the cytosolic fraction to the particulate fraction. Translocation of RhoA to the particulate fraction was observed within 15 min following stimulation of M1 mAChRs, indicating that RhoA is activated with sufficient rapidity to participate in the induction of zif 268 mRNA. Together, these results suggest that RhoA is activated following stimulation of M1 mAChRs and functions in SRE-dependent induction of the zif 268 gene within a Ras-independent pathway.

Calcium Signaling and c-Fos Gene Expression via M3 Muscarinic Acetylcholine Receptors in Human T- and B-Cells

We previously showed that blood acetylcholine (ACh) originates mainly from T-lymphocytes, and that stimulation of muscarinic ACh receptors (mAChRs) induces Ca2+ oscillations and up-regulates c-fos gene expression in both T- and B-lymphocytes. In the present study, we investigated which mAChR subtypes are involved in Ca2+ signaling and c-fos gene expression in human T- (CEM) and B- (Daudi) cells. Stimulation of mAChRs with 100 microM oxotremorine-M, an M1/M3 agonist, increased levels of intracellular free Ca2+ ([Ca2+]i) and c-fos mRNA expression in both cell lines. 4-DAMP, an M3 antagonist, more effectively blocked the oxotremorine-M-induced increase in [Ca2+]i than pirenzepine and telenzepine, M1-receptor antagonists; AF-DX 116, an M2 antagonist; hexahydrosiladifenidol, a weak M3 antagonist; or hexamethonium and d-tubocurarine, nicotinic receptor antagonists. McN-A-343 (100 microM), a partial M1-receptor agonist, had no apparent effect on [Ca2+]i in either cell line. The oxotremorine-M-induced up-regulation of c-fos transcription was inhibited by 4-DAMP, but not by pirenzepine or AF-DX 116. Our findings thus suggest that ACh released from T-lymphocytes acts as an autocrine/paracrine factor, transmitting a Ca2+-dependent signal to the nuclei of T- and B-lymphocytes via M3 receptors.

Differential cholinoceptor subtype-dependent activation of signal transduction pathways in neonatal versus adult rat atria

In this study, we investigated the expression and distribution of muscarinic cholinergic receptors (mAChRs) and the different signaling pathways associated with mAChR activation in atria isolated from adult and neonatal rats. Carbachol stimulation of mAChRs in both neonatal and adult rat atria led to a negative inotropic response with activation of phosphoinositide hydrolysis, an increase in cyclic GMP levels, and a decrease in cyclic AMP production. However, compared with adult atria, neonatal atria showed hypersensitivity in the contractile effect induced by carbachol. Pharmacological analysis with mAChR antagonists indicated that M 1 and M 2 mAChR subtypes are important mediators of the response to carbachol in neonatal atria. In contrast, in adult atria the effect of the agonist was coupled only to the M 2 mAChR subtype. Moreover, an increased number of total mAChRs was labeled in neonatal atrial membranes compared with those of adults. Although a predominant M 2 mAChR population is expressed in atria at both stages of development studied, competition binding parameters calculated for carbachol indicated the presence of high-affinity binding sites, with higher affinity in neonates than in adults. These results suggest that the differences observed between neonatal and adult atria in their response to a cholinergic agonist may be related to differential expression of mAChR subtypes and/or changes in functional coupling of mAChR subtypes during development.