mACh Receptor Research Articles

Effects of stimulation of muscarinic acetylcholine receptors in medial septum on some immune responses in rats

ObjectivesThough the immunomodulatory role of medial septum (MS) has been indicated, but the contribution of the muscarinic acetylcholine (mAch) receptors presented in the internal network of the neurons of MS in this regard is not known. The aim of the present study is to assess the contribution of mAch receptors of MS on some immunological parameters. MethodsDifferent immunological parameters i.e. phagocytic activity of peripheral leukocytes, adhesibility and cytotoxicity of splenic mononuclear cells (MNC), delayed type of hypersensitivity (DTH) responses, Total Count (TC) and Differential Count (DC) of WBC with serum corticosterone (CORT) concentration have been measured after stimulation and blocking of mAch receptors of MS in rats. Ach or atropine has been micro infused into MS of separate groups of rats for stimulating or blocking of the mAch receptors respectively. ResultsIn Ach or atropine microinfused rats, the TC of WBC remained unaltered in the present study. The percentage of neutrophil has increased and the percentage of lymphocyte has decreased in Ach microinfused rats, but these parameters remain unaltered in atropine microinfused rats. The observed immunological parameters have increased after microinfusion of 0.60μM and 0.12μM of Ach and serum CORT concentration has decreased in those animals. These immunological parameters have decreased and serum CORT has increased after microinfusion of atropine (2 and 4mM) in MS of rats. ConclusionIt appears that the Ach receptors in MS can modulate the observed immunological parameters, and serum CORT plays an important role for these immune changes.

Treatment with a muscarinic acetylcholine receptor antagonist impairs the acquisition of conditioned reward learning in rats

The neural mechanisms whereby a reward-associated stimulus gains reinforcing properties and comes to function as a conditioned reward (CR) are not understood. We propose that muscarinic acetylcholine (mACh) receptor stimulation is necessary for this type of learning. Here we tested the hypothesis that mACh receptor antagonism, with scopolamine, would attenuate the acquisition by a food-related stimulus of the capacity to function as a CR. Rats were exposed to 5 pre-exposure sessions during which two levers were present, one producing a light and the other a tone when pressed. This was followed by 3 conditioning sessions in which the levers were absent and the rats were presented with 30 light-food pairings delivered randomly. In the test session, the levers were present and presses on both levers were recorded. Different groups of rats received intraperitoneal injections of scopolamine (0, 0.375, 0.75 and 1mg/kg) either prior to each conditioning session or prior to the test session. All groups showed significantly greater responding on the light lever in the test compared to the pre-exposure sessions, demonstrating the CR effect. In animals treated prior to conditioning the scopolamine groups pressed significantly less on the light lever than the vehicle group. In animals treated prior to the test the increased lever pressing for light was similar for all groups. These data suggest that scopolamine impaired the acquisition of CR but not its expression. The results support the hypothesis that mACh receptor stimulation is important for the acquisition by reward-associated stimuli of the ability to function as CRs.

Rivastigmine improves isolation rearing-induced prepulse inhibition deficits via muscarinic acetylcholine receptors in mice.

The acetylcholinesterase inhibitors donepezil, galantamine, and rivastigmine are used for the treatment of Alzheimer's disease. We previously demonstrated that donepezil and galantamine differentially affect isolation rearing-induced prepulse inhibition (PPI) deficits and that this might be due to differential effects on brain muscarinic acetylcholine (mACh) receptor function in mice. We examined the effects of rivastigmine on isolation rearing-induced PPI deficits, brain ACh levels, and mACh receptor function in mice. Acoustic startle responses were measured in a startle chamber. Microdialysis was performed, and the levels of dopamine and ACh in the prefrontal cortex were measured. Rivastigmine (0.3 mg/kg) improved PPI deficits, and this improvement was antagonized by the mACh receptor antagonist telenzepine but not by the nicotinic ACh receptor antagonist mecamylamine. Rivastigmine increased extracellular ACh levels by approximately 2-3-fold, less than the increase produced by galantamine. Rivastigmine enhanced the effect of the mACh receptor agonist N-desmethylclozapine on prefrontal dopamine release, a marker of mACh receptor function, and this increase was blocked by telenzepine. In contrast, galantamine did not affect N-desmethylclozapine-induced dopamine release. Furthermore, rivastigmine did not affect cortical dopamine release induced by the serotonin1A receptor agonist osemozotan, suggesting that the effect of rivastigmine has specificity for mACh receptors. Taken together with our previous finding that marked increases in ACh levels are required for the PPI deficit improvement induced by galantamine, our present results suggest that rivastigmine improves isolation rearing-induced PPI deficits by increasing ACh levels and by concomitantly enhancing mACh receptor function.

Fasting stimulates 2-AG biosynthesis in the small intestine: role of cholinergic pathways.

The endocannabinoids are lipid-derived signaling molecules that control feeding and energy balance by activating CB1-type cannabinoid receptors in the brain and peripheral tissues. Previous studies have shown that oral exposure to dietary fat stimulates endocannabinoid signaling in the rat small intestine, which provides positive feedback that drives further food intake and preference for fat-rich foods. We now describe an unexpectedly broader role for cholinergic signaling of the vagus nerve in the production of the endocannabinoid, 2-arachidonoyl-sn-glycerol (2-AG), in the small intestine. We show that food deprivation increases levels of 2-AG and its lipid precursor, 1,2-diacylglycerol, in rat jejunum mucosa in a time-dependent manner. This response is abrogated by surgical resection of the vagus nerve or pharmacological blockade of small intestinal subtype-3 muscarinic acetylcholine (m3 mAch) receptors, but not inhibition of subtype-1 muscarinic acetylcholine (m1 mAch). We further show that blockade of peripheral CB1 receptors or intestinal m3 mAch receptors inhibits refeeding in fasted rats. The results suggest that food deprivation stimulates 2-AG-dependent CB1 receptor activation through a mechanism that requires efferent vagal activation of m3 mAch receptors in the jejunum, which, in turn, may promote feeding after a fast.

Synthesis and biological evaluation of isoxazoline derivatives as potent M₁ muscarinic acetylcholine receptor agonists.

A series of azacyclic compounds substituted with isoxazole and 5-substituted isoxazolines were synthesized as acyclic modifications of the oxime class M1 mACh receptor agonist. Among them, 3-(tetrahydropyrin-3-yl)-5-(2-pyrrolodin-1-yl)isoxazoline compound 4f displayed potent and selective M1 mACh receptor agonist activity in the functional calcium mobilization assay (EC50=31 nM). Introduction of 2-pyrrolidinone and 3-tetrahydropyridine groups are pivotal to the high potency. Moreover, 4f was found to facilitate non-amyloidogenic amyloid precursor protein (APP) processing by significantly increasing ERK1/2 phosphorylation and sAPPα secretion, known disease-modifying effects related to M1 mAChR agonists in Alzheimer's disease (AD).

Cholinergic modulation of primary afferent glutamatergic transmission in rat medullary dorsal horn neurons

Although muscarinic acetylcholine (mACh) receptors are expressed in trigeminal ganglia, it is still unknown whether mACh receptors modulate glutamatergic transmission from primary afferents onto medullary dorsal horn neurons. In this study, we have addressed the cholinergic modulation of primary afferent glutamatergic transmission using a conventional whole cell patch clamp technique. Glutamatergic excitatory postsynaptic currents (EPSCs) were evoked from primary afferents by electrical stimulation of trigeminal tract and monosynaptic EPSCs were recorded from medullary dorsal horn neurons of rat horizontal brain stem slices. Muscarine and ACh reversibly and concentration-dependently decreased the amplitude of glutamatergic EPSCs and increased the paired-pulse ratio. In addition, muscarine reduced the frequency of miniature EPSCs without affecting the current amplitude, suggesting that muscarine acts presynaptically to decrease the probability of glutamate release onto medullary dorsal horn neurons. The muscarine-induced decrease of glutamatergic EPSCs was significantly occluded by methoctramine or AF-DX116, M2 receptor antagonists, but not pirenzepine, J104129 and MT-3, selective M1, M3 and M4 receptor antagonists. The muscarine-induced decrease of glutamatergic EPSCs was highly dependent on the extracellular Ca2+ concentration. Physostigmine and clinically available acetylcholinesterase inhibitors, such as rivastigmine and donepezil, significantly shifted the concentration–inhibition relationship of ACh for glutamatergic EPSCs. These results suggest that muscarine acts on presynaptic M2 receptors to inhibit glutamatergic transmission by reducing the Ca2+ influx into primary afferent terminals, and that M2 receptor agonists and acetylcholinesterase inhibitors could be, at least, potential targets to reduce nociceptive transmission from orofacial tissues.

Patterned high-frequency stimulation induces a form of long-term depression dependent on GABAA and mACh receptors in the hippocampus

Certain patterns of neural activity can induce N-methyl-d-aspartic acid receptor (NMDAR)-dependent synaptic plasticity, one of the important foundations of memory. Here, we report that a patterned high-frequency stimulation (PHS) induces rat hippocampal long-term depression (LTD) in an NMDAR-independent manner that requires coactivation of GABAARs and muscarinic acetylcholine receptors (mAChRs), and endocytosis of AMPARs. Thus, we disclose that a patterned high-frequency stimulation triggers GABAAR and mAChR-dependent LTD in the hippocampus.

Muscarinic M4 receptors regulate GABAergic transmission in rat tuberomammillary nucleus neurons

Histaminergic neurons within the tuberomammillary nucleus (TMN) play an important role in sleep-wakefulness regulation. Here, we report the muscarinic modulation of GABAergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in mechanically dissociated rat histaminergic neurons using a conventional whole-cell patch clamp technique. Muscarine, a nonselective muscarinic acetylcholine (mACh) receptor agonist, reversibly decreased mIPSC frequency without affecting the current amplitude, indicating that muscarine acts presynaptically to decrease the probability of spontaneous GABA release. The muscarine action on GABAergic mIPSC frequency was completely blocked by atropine, a nonselective mACh receptor antagonist, and tropicamide, an M4 receptor antagonist. The muscarine-induced decrease in mIPSC frequency was completely occluded in the presence of Cd2+, a general voltage-dependent Ca2+ channel blocker, or in a Ca2+-free external solution. However, pharmacological agents affecting adenylyl cyclase or G-protein coupled inwardly rectifying K+ channel activity did not prevent the inhibitory action of muscarine on GABAergic mIPSCs. These results suggest that muscarine acts on M4 receptors on GABAergic nerve terminals projecting to histaminergic neurons to inhibit spontaneous GABA release via the inhibition of Ca2+ influx from the extracellular space. Muscarine also inhibited action potential-dependent GABA release by activating presynaptic M4 receptors in more physiological conditions. The M4 receptor-mediated modulation of GABAergic transmission onto TMN neurons may contribute to the regulation of sleep-wakefulness.

Continued optimization of the MLPCN probe ML071 into highly potent agonists of the hM1 muscarinic acetylcholine receptor

This Letter describes the continued optimization of the MLPCN probe molecule ML071. After introducing numerous cyclic constraints and novel substitutions throughout the parent structure, we produced a number of more highly potent agonists of the M1 mACh receptor. While many novel agonists demonstrated a promising ability to increase soluble APPα release, further characterization indicated they may be functioning as bitopic agonists. These results and the implications of a bitopic mode of action are presented.

Neurotransmitter Influence on Human Meibomian Gland Epithelial Cells

A striking characteristic of the human meibomian gland is its rich sensory, sympathetic, and parasympathetic innervation, yet the functional relevance of these nerve fibers remains unknown. Acting on the hypothesis that neurotransmitters are released in the vicinity of the gland, act on glandular receptors, and influence the production, secretion, and/or delivery of meibomian gland secretions to the ocular surface, the goal in this study was to begin to determine whether neurotransmitters influence the meibomian gland. Immortalized human meibomian gland epithelial (SLHMG) cells were examined for the presence of vasoactive intestinal peptide (VIP) and muscarinic acetylcholine (mACh) receptor transcripts and proteins. Cells were also exposed to VIP, carbachol, forskolin, and/or 3-isobutyl-1-methylxanthine (IBMX) to determine whether these agents, alone or in combination, modulate the adenylyl cyclase pathway, the accumulation of intracellular free calcium ([Ca2+]i), or cell proliferation. Results demonstrate that SLHMG cells transcribe and translate VIP and mACh receptors; VIP, with either IBMX or forskolin, activates the adenylyl cyclase pathway, and the effect of VIP and forskolin together is synergistic; both VIP and carbachol increase intracellular [Ca2+] in SLHMG cells; and VIP with forskolin stimulates SLHMG cell proliferation. This study shows that parasympathetic neurotransmitters and their agonists influence the function of human meibomian gland epithelial cells. It remains to be determined whether this action alters the production, secretion, and/or delivery of meibum to the ocular surface.

Synthesis of 99mTc-oxybutynin for M3-receptor-mediated imaging of urinary bladder

Radiolabeling of oxybutynin, a muscarinic acetylcholine (mACh) receptor antagonist agent with 99mTc is of considerable interest for imaging of urinary bladder. This study is aimed to optimize radiolabeling yield of oxybutynin with 99mTc using SnCl2·2H2O as a reducing agent with respect to factors that affect the reaction conditions such as oxybutynin amount, stannous chloride amount, reaction time and pH of the reaction mixture. In vitro stability of the radiolabeled complex was checked and it was found to be stable for up to 8 h. 99mTc-oxybutynin was injected via subcutaneous and intravenous administration routes into normal Sprague–Dawley rats. Biodistribution studies have revealed that 99mTc-oxybutynin exhibits high affinity and specificity for the muscarinic M3 subtype located on the smooth muscle of urinary bladder relative to the M1 and M2 subtypes of the G protein coupled receptor (GPCR) superfamily. In vivo uptake of subcutaneous 99mTc-oxybutynin in urinary bladder was 19.6 ± 0.42% ID at 0.5 h, whereas in intravenous administration route the accumulation in the urinary bladder was found to be 9.4 ± 0.31% ID at 0.5 h post injection. Administration of cold oxybutynin effectively blocked urinary bladder uptake and further confirms the high specificity of this complex for the M3 receptor.

Contrasting Effects of Allosteric and Orthosteric Agonists on M1Muscarinic Acetylcholine Receptor Internalization and Down-regulation

A new class of subtype-selective muscarinic acetylcholine (mACh) receptor agonist that activates the receptor through interaction at a site distinct from the orthosteric acetylcholine binding site has been reported recently. Here, we have compared the effects of orthosteric (oxotremorine-M, arecoline, pilocarpine) and allosteric [4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine (AC-42); 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1)] agonists on M(1) mACh receptor internalization and down-regulation, as well as functional coupling in a Chinese hamster ovary (CHO) cell line. In contrast to full and partial orthosteric agonists, which cause significant receptor internalization and down-regulation, prolonged exposure to AC-42 did not significantly alter either cell-surface or total cellular M(1) mACh receptor expression. 77-LH-28-1, an AC-42 homolog, did cause some receptor internalization, but not down-regulation. The presence of atropine completely prevented the orthosteric agonist-induced adaptive changes in receptor populations; however, in contrast, the copresence of atropine and AC-42 significantly increased both cell-surface receptor and total M(1) mACh receptor expression. Maximal phosphoinositide hydrolysis responses to the partial agonist arecoline were similar in CHO-M(1) cells pretreated for 24 h with either AC-42 or vehicle; in contrast, these responses were markedly reduced when cells were pretreated with oxotremorine-M or pilocarpine. These data indicate that, whereas AC-42 binding to the M(1) mACh receptor can initiate signal transduction, the AC-42-liganded receptor is resistant to the usual mechanisms regulating receptor internalization and down-regulation. In addition, our data suggest unusual interactions between allosteric agonists and orthosteric antagonists to regulate cell-surface and total cellular receptor expression.

Effects of systemic phencyclidine on firing activity of ventral tegmental area neurons in a classical conditioning paradigm

The neural mechanisms whereby a reward-associated stimulus gains reinforcing properties and comes to function as a conditioned reward (CR) are not understood. We propose that muscarinic acetylcholine (mACh) receptor stimulation is necessary for this type of learning. Here we tested the hypothesis that mACh receptor antagonism, with scopolamine, would attenuate the acquisition by a food-related stimulus of the capacity to function as a CR. Rats were exposed to 5 pre-exposure sessions during which two levers were present, one producing a light and the other a tone when pressed. This was followed by 3 conditioning sessions in which the levers were absent and the rats were presented with 30 light-food pairings delivered randomly. In the test session, the levers were present and presses on both levers were recorded. Different groups of rats received intraperitoneal injections of scopolamine (0, 0.375, 0.75 and 1 mg/kg) either prior to each conditioning session or prior to the test session. All groups showed significantly greater responding on the light lever in the test compared to the pre-exposure sessions, demonstrating the CR effect. In animals treated prior to conditioning the scopolamine groups pressed significantly less on the light lever than the vehicle group. In animals treated prior to the test the increased lever pressing for light was similar for all groups. These data suggest that scopolamine impaired the acquisition of CR but not its expression. The results support the hypothesis that mACh receptor stimulation is important for the acquisition by reward-associated stimuli of the ability to function as CRs.

G Protein Coupling and Signaling Pathway Activation by M1Muscarinic Acetylcholine Receptor Orthosteric and Allosteric Agonists

The M(1) muscarinic acetylcholine (mACh) receptor is among a growing number of G protein-coupled receptors that are able to activate multiple signaling cascades. AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine) is an allosteric agonist that can selectively activate the M(1) mACh receptor in the absence of an orthosteric ligand. Allosteric agonists have the potential to stabilize unique receptor conformations, which may in turn cause differential activation of signal transduction pathways. In the present study, we have investigated the signaling pathways activated by AC-42, its analog 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone), and a range of orthosteric muscarinic agonists [oxotremorine-M (oxo-M), arecoline, and pilocarpine] in Chinese hamster ovary cells recombinantly expressing the human M(1) mACh receptor. Each agonist was able to activate Galpha(q/11)-dependent signaling, as demonstrated by an increase in guanosine 5'-O-(3-thiotriphosphate) ([(35)S]GTPgammaS) binding to Galpha(q/11) proteins and total [(3)H]inositol phosphate accumulation assays in intact cells. All three orthosteric agonists caused significant enhancements in [(35)S]GTPgammaS binding to Galpha(i1/2) subunits over basal; however, neither allosteric ligand produced a significant response. In contrast, both orthosteric and allosteric agonists are able to couple to the Galpha(s)/cAMP pathway, enhancing forskolin-stimulated cAMP accumulation. These data provide support for the concept that allosteric and orthosteric mACh receptor agonists both stabilize receptor conformations associated with Galpha(q/11)- and Galpha(s)-dependent signaling; however, AC-42 and 77-LH-28-1, unlike oxo-M, arecoline, and pilocarpine, do not seem to promote M(1) mACh receptor-Galpha(i1/2) coupling, suggesting that allosteric agonists have the potential to activate distinct subsets of downstream effectors.

Differential Regulation of the M1 Muscarinic Acetylcholine Receptor by Orthosteric and Allosteric Ligands

AC‐42 (4‐n‐butyl‐1‐[4‐(2‐methylphenyl)‐4‐oxo‐1‐butyl] piperidine hydrogen chloride) is a selective M1 muscarinic receptor agonist with an allosteric mechanism of action. Here, we have investigated whether AC‐42 and two orthosteric muscarinic agonists (oxotremorine‐M (oxo‐M) and pilocarpine) cause internalization and down‐regulation of the M1 muscarinic receptor. Oxo‐M (EC50 0.8 μM) caused the greatest increase in [35S]‐GTPγS‐Gαq/11 binding in membranes from Chinese hamster ovary cells stably expressing the M1 muscarinic receptor, whereas AC‐42 (EC50 1.6 μM) and pilocarpine (EC50 2.4 μM) stimulated similar responses that were approx. 30% of the oxo‐M response. Using intact cell [3H]‐NMS and [3H]‐QNB binding assays, we next showed that after 24 h, oxo‐M (100 ìM) caused 68 ± 3% receptor internalization and 42 ± 3% receptor down‐regulation. Over a similar time‐course, pilocarpine (1 mM) caused 46 ± 7% internalization and 26 ± 8% down‐regulation. In contrast, AC‐42 did not cause significant receptor internalization or down‐regulation. These data suggest that while AC‐42 is able to activate selectively Gq/11‐dependent signaling through the M1 mACh receptor, it does not cause receptor internalization or down‐regulation, at least over the time‐course studied here.

The effects of mercury on muscarinic cholinergic receptor subtypes (M1 and M2) in captive mink

A combination of in vitro (competitive binding assays) and in vivo (tissues from animals exposed to dietary methyl mercury, MeHg) experimental procedures was employed to assess the effects of mercury (MeHg, HgCl 2) on the two-key muscarinic cholinergic (mACh) receptor subtypes (M1, M2) in two brain regions (occipital cortex, brain stem) of captive mink ( Mustela vison). In vitro, HgCl 2 and MeHg were equipotent in inhibiting [ 3H]-pirenzipine binding to the M1 receptor in the occipital cortex, but in the brain stem, MeHg was about 65× more potent than HgCl 2. For the M2 receptor, both HgCl 2 and MeHg were more potent at inhibiting [ 3H]-AFDX-384 binding in the occipital cortex than in the brain stem. Within each brain region, HgCl 2 was more potent at inhibiting [ 3H]-AFDX-384 binding than MeHg. In vivo exposure of captive mink to MeHg (0.5, 1, and 2 ppm MeHg in the diet for 89 days) resulted in greater binding of radioligands to the M1 and M2 receptor in the occipital cortex, but not in the brain stem, when compared to control animals. Based on the in vitro results, we could not conclude which mACh receptor subtype or brain region was most sensitive to Hg, but the in vivo findings suggest that Hg preferentially affects mACh receptor subtype (M1 and M2) levels in the occipital cortex. By studying distinct mACh receptors, these results extend upon previous studies in laboratory rodents and wildlife that showed Hg to affect the global population of mACh receptors.

Regulation of cyclic AMP response-element binding-protein (CREB) by Gq/11-protein-coupled receptors in human SH-SY5Y neuroblastoma cells

Human SH-SY5Y neuroblastoma cells have been used to investigate mechanisms involved in CREB phosphorylation after activation of two endogenously expressed Gq/11-protein-coupled receptors, the M3 muscarinic acetylcholine (mACh) and B2 bradykinin receptors. Stimulation with either methacholine or bradykinin resulted in maximal increases in CREB phosphorylation within 1min, with either a rapid subsequent decrease (bradykinin) to basal levels, or a sustained response (methacholine). Inhibitor studies were performed to assess the involvement of a number of potential kinases in signalling to CREB phosphorylation. Removal of extracellular Ca2+, inhibition of Ca2+/calmodulin-dependent protein kinase II and down-regulation of protein kinase C (PKC) resulted in reduced CREB phosphorylation after both M3 mACh and B2 bradykinin receptor activation. In contrast, inhibition of MEK1/2 by U0126 resulted in significantly reduced CREB phosphorylation levels after B2 bradykinin, but not M3 mACh receptor activation. In addition, we demonstrate that maintained phosphorylation of CREB is necessary for CRE-dependent gene transcription as the M3 mACh, but not the B2 bradykinin receptor activates both a recombinant CRE-dependent reporter gene, and the endogenous c-Fos gene. These data highlight the involvement of multiple, overlapping signalling pathways linking these endogenous Gq/11-coupled metabotropic receptors to CREB and emphasize the importance of the duration of signalling pathway activation in converting a CREB phosphorylation event into a significant change in transcriptional activity.

The regulation of M1 muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons1

To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC50 concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M1 mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABAA receptors with picrotoxin. The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade. Inhibition of endogenous G protein-coupled receptor kinase 2 by transfection with the non-Gq/11α-binding, catalytically-inactive D110A,K220RG protein-coupled receptor kinase 2 mutant, decreased the extent of M1 mACh receptor desensitization under all conditions. Pharmacological inhibition of protein kinase C (PKC) activity, or chronic phorbol ester-induced PKC down-regulation had no effect on agonist-mediated receptor desensitization in quiescent or spontaneously synaptically active neurons, but significantly decreased the extent of receptor desensitization in picrotoxin-treated neurons. MCh stimulated the translocation of diacylglycerol- sensitive eGFP-PKCε, but not Ca2+/diacylglycerol-sensitive eGFP-PKCβII in both the absence, and presence of tetrodotoxin. Under these conditions, MCh-stimulated eGFP-myristoylated, alanine-rich C kinase substrate translocation was dependent on PKC activity, but not Ca2+/calmodulin. In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCβII, but not PKCε, and was dependent on PKC and Ca2+/calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons.

In vivo investigations on the cholinesterase-inhibiting effects of tricyclic quinazolinimines: Scopolamine-induced cognitive impairments in rats are attenuated at low dosage and reinforced at higher dosage

Tricyclic quinazolinimines as a novel class of potent inhibitors of cholinesterases in vitro are micro- and sub-micromolar inhibitors with activities at both acetyl- (AChE) and butyrylcholinesterase (BChE) or at BChE only. To further establish the antiamnesic properties of this class of compounds, an in vivo test system has been established. Cognitive impairment in rats was reversibly induced by scopolamine (0.05 mg/100 g body weight) and evaluated in an eight-arm radial maze. A representative quinazolinimine (MD212) showed attenuation of cognitive deficits at a low dosage (0.01 mg/100 g body weight), whereas at a high dosage (>0.1 mg/100 g body weight) the effect of scopolamine is markedly reinforced. As MD212 applied alone does not influence rat's cognition at all, the reinforcement of scopolamine effect has to be due to the amplification of scopolamine action possibly by (1) inhibition of scopolamine metabolism, (2) influence of scopolamine on MD212 metabolism or (3) allosteric modulation of mACh receptors. Receptor-binding studies proved hypothesis (3): MD212 stabilizes [3H]N-methylscopolamine binding to muscarinic receptors allosterically.

Expression of mRNA encoding G protein-coupled receptors involved in congestive heart failure

Congestive heart failure (CHF) induces changes in the neurohumoral system and gene expression in viable myocardium. Several of these genes encode G protein-coupled receptors (GPCRs) involved in mechanisms which compensate for impaired myocardial function. We used real-time quantitative RT-PCR (Q-RT-PCR) to investigate the expression of mRNA encoding 15 different GPCRs possibly involved in CHF, and the effect of normalisation to GAPDH mRNA (GAPDH) or 18S rRNA (18S). CHF was induced in rats by coronary artery ligation, with sham-operated controls (Sham). After 6 weeks, mRNA expression in viable left ventricular myocardium was determined using both 18S and GAPDH as the normalisation standard. An apparent 30% reduction in GAPDH mRNA levels vs. 18S in CHF compared to Sham, although not significant in itself, influenced the interpretation of regulation of other genes.Thus, levels of mRNA encoding receptors for angiotensin II (AT(1)), endothelin (ET(A), ET(B)) and the muscarinic acetylcholine (mACh) receptor M(1) increased significantly in CHF only when normalised to GAPDH. Levels of mRNA encoding the mACh receptors M(3) and M(4) and the serotonin receptors 5-HT(2A) and 5-HT(4) increased, whereas alpha(1D)-adrenoceptor mRNA decreased in CHF irrespective of the normalisation standard. No significant change was detected for M2 and M5 mACh receptors or alpha(1A)-, alpha(1B)-, beta(1)- or beta(2)-adrenoceptors. Q-RT-PCR is a sensitive and powerful method to monitor changes in GPCR mRNA expression in CHF. However, the normalisation standard used is important for the interpretation of mRNA regulation.