Changes in rat brain muscarinic receptors after inhibitory avoidance learning

It is widely accepted that cerebral acetylcholine is necessary for learning and memory, but little is known about the type of muscarinic receptors involved in these functions. To investigate this problem, [3H]-N-methyl-scopolamine which binds to different types of muscarinic receptors, [3H]-Pirenzepine an M1 receptor antagonist, and [3H]-Oxotremorine-M which binds mainly to M2 receptors, were used as ligands to look for possible changes in muscarinic receptor density in neostriatum (NEO), hippocampus (HIP), amygdala (AMY), and temporo-parietal neocortex (CTX), after testing for retention of inhibitory avoidance, trained with high or low footshock intensities. After low reinforcement there was an M1 postsynaptic receptor up-regulation in NEO, HIP, and CTX, and an M2 presynaptic receptor down-regulation in HIP, which suggests a concerted pre- and postsynaptic cholinergic activation in this area. An up-regulation of both M1 and M2 receptors was detected in CTX of low and high footshocked animals, which indicates the presence of a cortical postsynaptic M2 receptor.     

Differential regulation of expression of rat hippocampal muscarinic receptor subtypes following fimbria-fornix lesion

Quantitative RNase protection assays were performed to determine the levels of muscarinic receptor subtype (m1-m5) mRNAs in rat hippocampi. Results showed that the m1, m3, and m4 subtype mRNAs were expressed at relatively high levels, but the levels of the m2 and m5 subtype were very low. Three weeks following aspiration lesions of the fimbria-fornix to produce cholinergic denervation of the hippocampus, non-M1 receptors (non-pirenzepine displaceable [3H]quinuclindinyl benzilate binding sites) in the hippocampus were increased significantly, which correlated with increases in the levels of hippocampal m3 and m4 receptor mRNAs (m3: +24% and m4: +41%). These findings indicate that multiple muscarinic receptor subtypes are expressed in the hippocampus with the m3 and m4 subtypes predominantly postsynaptic to the septohippocampal cholinergic terminals.     

Pharmacological characterization of the novel cholinomimetic L-689,660 at cloned and native brain muscarinic receptors

1-Azabicyclo[2,2,2]octane,3-(6-chloropyrazinyl)maleate (L-689,660) reportedly is an agonist with selectivity for M1 and M3 muscarinic receptors. We confirmed this in functional assays of brain muscarinic receptors and of cloned human muscarinic receptors transfected into Chinese hamster ovary (CHO-K1) cells. For stimulation of phosphoinositide turnover in rat cortical and hippocampal dissociated tissue, L-689,660 was a partial agonist (24% and 26% intrinsic activity, respectively, relative to oxotremorine-M) with EC50 values of 71 microM and 118 microM, respectively. At putative M4 receptors coupled to cyclic AMP inhibition in rat striatum, however, L-689,660 acted as a competitive antagonist (KB = 0.4 microM). Furthermore, at putative M2/M4 autoreceptors that regulate acetylcholine release in the hippocampus, the drug also behaved as an antagonist (KB = 2.1 microM). These data indicated that L-689,660 behaves as a postsynaptic agonist/presynaptic antagonist at central cholinergic synapses. Further aspects of the selectivity of the drug for specific muscarinic receptor subtypes were revealed with phosphoinositide turnover assays of cloned muscarinic receptors expressed in CHO-K1 cells. L-689,660 was a partial agonist at transfected hm1 and hm3 receptors and was more potent than oxotremorine-M; however, the drug was inactive at transfected hm5 receptors. Partial agonist activity at hm1 and hm3 muscarinic receptors was present even after using alkylation to reduce receptor numbers to levels comparable to that level found in the hm5 cell line. Thus, with functional assays either with brain tissue or with transfected cell lines, L-689,660 was shown to be an agonist for the M1 and M3 receptors but not for M5 or M4 receptors.     

Differential involvement of group II and group III mGluRs as autoreceptors at lateral and medial perforant path synapses

1. Previous reports have shown that group III metabotropic glutamate receptors (mGluRs) serve as autoreceptors at the lateral perforant path, but to date there has been no rigorous determination of the roles of other mGluRs as autoreceptors at this synapse. Furthermore, it is not known which of the mGluR subtypes serve as autoreceptors at the medial perforant path synapse. With the use of whole cell patch-clamp and field excitatory postsynaptic potential (fEPSP) recording techniques, we examined the groups of mGluRs that act as autoreceptors at lateral and medial perforant path synapses in adult rat hippocampal slices. 2. Consistent with previous reports, the group III mGluR agonist (D,L)-2-amino-4-phosphonobutyric acid reduced fEPSPs and excitatory postsynaptic currents (EPSCs) in the dentate gyrus. However, the group-II-selective agonist (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) also reduced fEPSPs and EPSCs, suggesting that multiple mGluR subtypes may serve as autoreceptors at perforant path synapses. 3. Selective activation of either medial or lateral perforant pathways revealed that micromolar concentrations of (L)-2-amino-4-phosphonobutyric acid (L-AP4) reduce fEPSPs in lateral but not medial perforant path, suggesting group III involvement at the lateral perforant pathway. Conversely, DCG-IV and 2R, 4R-4-aminopyrrolidine-2,4-dicarboxylate, another group-II-selective mGluR agonist, potently reduced fEPSPs at the medial but not lateral perforant path, suggesting that a group II mGluR may act as an autoreceptor at the medial perforant path-dentate gyrus synapse. 4. Antagonist studies with group-selective antagonists such as (2S,3S,4S)-2-methyl-2-(carboxycyclpropyl)glycine (MCCG; group II) and alpha-methyl-L-AP4 (MAP4; group III) suggest differential involvement of each group at these synapses. The effect of L-AP4 at the lateral perforant path synapse was blocked by MAP-4, but not MCCG. In contrast, the effect of DCG-IV was blocked by application of MCCG, but not MAP4. 5. Previous studies suggest that the effect of L-AP4 at the lateral perforant path synapse is mediated by a presynaptic mechanism. In the present studies, we found that concentrations of DCG-IV that reduce transmission at the medial perforant path synapse reduce paired-pulse depression and do not reduce kainate-evoked currents recorded from dentate granule cells. This is consistent with the hypothesis that DCG-IV also acts by a presynaptic mechanism.     

Subcellular redistribution of m2 muscarinic acetylcholine receptors in striatal interneurons in vivo after acute cholinergic stimulation

The purpose of our work was to investigate how the cholinergic environment influences the targeting and the intracellular trafficking of the muscarinic receptor m2 (m2R) in vivo. To address this question, we have used immunohistochemical approaches at light and electron microscopic levels to detect the m2R in control rats and rats treated with muscarinic receptor agonists. In control animals, m2Rs were located mostly at postsynaptic sites at the plasma membrane of perikarya and dendrites of cholinergic and NPY-somatostatin interneurons as autoreceptors and heteroreceptors, respectively. Presynaptic receptors were also detected in boutons. The m2Rs were usually detected at extrasynaptic sites, but they could be found rarely in association with symmetrical synapses, suggesting that the cholinergic transmission mediated by m2R occurs via synaptic and nonsynaptic mechanisms. The stimulation of muscarinic receptors with oxotremorine provoked a dramatic alteration of m2R compartmentalization, including endocytosis with a decrease of the density of m2R at the membrane (-63%) and an increase of those associated with endosomes (+86%) in perikarya. The very strong increase of m2R associated with multivesicular bodies (+732%) suggests that oxotremorine activated degradation. The slight increase in the Golgi apparatus (+26%) suggests that the m2R stimulation had an effect on the maturation of m2R. The substance P receptor located at the membrane of the same neurons was unaffected by oxotremorine. Our data demonstrate that cholinergic stimulation dramatically influences the subcellular distribution of m2R in striatal interneurons in vivo. These events may have key roles in controlling abundance and availability of muscarinic receptors via regulation of receptor endocytosis, degradation, and/or neosynthesis. Further, the control of muscarinic receptor trafficking may influence the activity of striatal interneurons, including neurotransmitter release and/or electric activity.     

Activation of the genetically defined m1 muscarinic receptor potentiates N-methyl-D-aspartate (NMDA) receptor currents in hippocampal pyramidal cells

Evidence suggests that cholinergic input to the hippocampus plays an important role in learning and memory and that degeneration of cholinergic terminals in the hippocampus may contribute to the memory loss associated with Alzheimer's disease. One of the more prominent effects of cholinergic agonists on hippocampal physiology is the potentiation of N-methyl-D-aspartate (NMDA)-receptor currents by muscarinic agonists. Here, we employ traditional pharmacological reagents as well as m1-toxin, an m1 antagonist with unprecedented selectivity, to demonstrate that this potentiation of NMDA-receptor currents in hippocampal CA1 pyramidal cells is mediated by the genetically defined m1 muscarinic receptor. Furthermore, we demonstrate the colocalization of the m1 muscarinic receptor and the NR1a NMDA receptor subunit at the electron microscopic level, indicating a spatial relationship that would allow for physiological interactions between these two receptors. This work demonstrates that the m1-muscarinic receptor gene product modulates excitatory synaptic transmission, and it has important implications in the study of learning and memory as well as the design of drugs to treat neurodegenerative diseases such as Alzheimer's.     

Allosteric interactions of quaternary strychnine and brucine derivatives with muscarinic acetylcholine receptors

The affinity and allosteric properties of 22 quaternary derivatives of strychnine and brucine at the m1-m4 subtypes of muscarinic receptors have been analyzed and compared. The subtype selectivity, in terms of affinity, was in general m2 > m4 > m1 > m3. The highest affinities were found for N-benzyl, N-2-naphthylmethyl, and N-4-biphenylylmethyl strychnine (13, 14, and 18, respectively). All the strychnine and brucine derivatives were positively cooperative with the antagonist, N-methylscopolamine, at m2 receptors and, in the case of the strychnine analogues, were positively cooperative with N-methylscopolamine at least at one other subtype. The strychnine analogues were negatively cooperative with the neurotransmitter, acetylcholine, at all subtypes whereas brucine and five of the six derivatives examined were positively cooperative with acetylcholine at one or more subtypes (m1-m5) and exhibited different patterns of subtype selectivity. The ability to generate subtype-selective allosteric enhancers of acetylcholine binding and function may be of use in the development of drugs for the treatment of Alzheimer's disease.     

Muscarinic receptor subtypes in the lateral geniculate nucleus: a light and electron microscopic analysis

Neural activity in the dorsal lateral geniculate nucleus of the thalamus (DLG) is modulated by an ascending cholinergic projection from the brainstem. The purpose of this study was to identify and localize specific muscarinic receptors for acetylcholine in the DLG. Receptors were identified in rat and cat tissue by means of antibodies to muscarinic receptor subtypes, ml-m4. Brain sections were processed immunohistochemically and examined with light and electron microscopy. Rat DLG stained positively with antibodies to the m1, m2,and m3 receptor subtypes but not with antibodies to the m4 receptor subtype. The m1 and m3 antibodies appeared to label somata and dendrites of thalamocortical cells. The m1 immunostaining was pale, whereas m3-positive neurons exhibited denser labeling with focal concentrations of staining. Strong immunoreactivity to the m2 antibody was widespread in dendrites and somata of cells resembling geniculate interneurons. Most m2-positive synaptic contacts were classified as F2-type terminals, which are the presynaptic dendrites of interneurons. The thalamic reticular nucleus also exhibited robust m2 immunostaining. Cat DLG exhibited immunoreactivity to the m2 and m3 antibodies. The entire DLG stained darkly for the m2 receptor subtype, except for patchy label in the medial interlaminar nucleus and the ventralmost C laminae. The staining for m3 was lighter and was distributed more homogeneously across the DLG. The perigeniculate nucleus also was immunoreactive to the m2 and m3 subtype-specific antibodies. Immunoreactivity in cat to the m1 or m4 receptor antibodies was undetectable. These data provide anatomical evidence for specific muscarinic-mediated actions of acetylcholine on DLG thalamocortical cells and thalamic interneurons.     

Activation of acetylcholine receptors and 5-HT2 receptors have additive effects in the suppression of neocortical high-voltage spindles in aged rats

We investigated if activation of the muscarinic or nicotinic acetylcholine receptors and serotonin (5-hydroxytryptamine; 5-HT) subtype 2 receptors would have additive or synergistic effects on the suppression of thalamocortically generated rhythmic neocortical high-voltage spindles (HVSs) in aged rats. The 5-HT2 receptor antagonist, ketanserin, at a moderate dose (5 mg/kg) prevented the ability of a muscarinic acetylcholine receptor agonist, (oxotremorine 0.1 mg/kg), and a nicotinic acetylcholine receptor agonist (nicotine 0.1 mg/kg), to decrease HVSs. At a higher dose (20 mg/kg), ketanserin completely blocked the decrease in HVSs produced by moderate doses of muscarinic acetylcholine receptor agonists (pilocarpine 1 mg/kg and oxotremorine 0.1 mg/kg), and by a high dose of nicotine (0.3 mg/kg), though not that produced by high doses of pilocarpine (3 mg/kg) and oxotremorine (0.9 mg/kg). The ability of a 5-HT2 receptor agonist, (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (0.1-1.0 mg/kg), to suppress HVSs was non-significantly modulated by the nicotinic acetylcholine receptor antagonist, mecamylamine (1-15 mg/kg), and the muscarinic acetylcholine receptor antagonist, scopolamine (0.03-0.3 mg/kg). The effects of the drugs on behavioral activity could be separated from their effects on HVSs. The results suggest that activation of the muscarinic or nicotinic acetylcholine receptors plus 5-HT2 receptors has additive effects in the suppression of thalamocortical oscillations in aged rats.     

RGS proteins reconstitute the rapid gating kinetics of gbetagamma-activated inwardly rectifying K+ channels

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Galpha(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20-40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of "regulators of G protein signaling" (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor --> GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent "basal" GIRK current was suppressed by approximately 40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of "slow" postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.     

Construction and overexpression in Escherichia coli of genetically engineered derivatives of penicillin-binding protein 2'' of Staphylococcus epidermidis

This paper reports a study of long-term potentiation (LTP) of perforant path synapses in CA1. Using rat hippocampal slices with CA3 and the dentate gyrus removed, stimulation of the perforant path evoked a population excitatory postsynaptic potential (pEPSP) that was negative-going in s. lacunosum-moleculare of CA1. High-frequency conditioning stimulation of the perforant pathway induced LTP of the perforant path pEPSP in slices disinhibited by the GABAA receptor antagonist bicuculline methiodide (20 microM). Conditioning of the perforant pathway in normal medium, however, failed to induce LTP. Potentiation of the perforant path pEPSP in the presence of bicuculline lasted at least 1 h, was specific to the tetanized pathway, and based on a threshold property, appeared associative in nature.     

Electrophysiological and pharmacological characterization of the direct perforant path input to hippocampal area CA3

Monosynaptic perforant path responses evoked by subicular stimulation were recorded from CA3 pyramidal cells of rat hippocampal slices. These monosynaptic responses were isolated by using low intensities of stimulation and by placing a cut through the mossy fibers. Perforant path-evoked responses consisted of both excitatory and inhibitory components. Excitatory postsynaptic currents (EPSCs) were mediated by both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acidreceptors (AMPAR) and N-methyl--aspartate receptors (NMDAR). Inhibitory postsynaptic currents consisted of gamma-aminobutyric acid-A (GABAA-) and -B (GABAB)-receptor-mediated components. At membrane potentials more positive than -60 mV and at physiological [Ca2+]/[Mg2+] ratios, >30% of perforant path evoked EPSC was mediated by NMDARs. This value varied as a function of the membrane voltage and external [Mg2+]. Two types of responses were observed after low-intensity stimulation of the perforant path. The first type of response showed paired-pulse facilitation and was reduced by 2-amino-4-phosphonobutyric acid (AP4). The second type of response showed paired-pulse depression and was reduced by baclofen. Electrophysiological and pharmacological characteristics of these two types of responses are similar to the properties of lateral and medial perforant path-evoked EPSPs in the dentate gyrus.     

Muscarinic acetylcholine receptor subtypes mediating urinary bladder contractility and coupling to GTP binding proteins

Activation of muscarinic acetylcholine receptors is primarily responsible for urinary bladder emptying. Because multiple subtypes of muscarinic receptors exist, we wished to characterize those present in bladder and ultimately to attribute function to those that regulate bladder contractility, neurotransmitter release and perhaps other cholinergic functions in this tissue. Although the m2 and m3 subtypes could be immunoprecipitated after solubilization from human, rat, rabbit and guinea pig bladder membranes, the m1, m4 and m5 subtypes could not. The m2:m3 ratio was 9:1 in rat bladder but was only 3:1 in the other species examined. Immunoprecipitation of the m2 subtype correlated with the relative levels of high-affinity agonist binding sites measured by competition of carbachol for [3H]N-methylscopolamine binding or measured directly using [3H]oxotremorine-M. In the presence of agonist, but not antagonist, GTP binding proteins could be immunoprecipitated in concert with the m2 or m3 receptors using anti-receptor antibodies. These proteins were members of the Gi and Gq/11 subfamilies for both the m2 and the m3 receptor subtypes. In spite of the preponderance of the m2 receptor in all species studied, Schild analysis using somewhat selective antagonists showed that the pharmacologically defined m3 receptor mediated contractility in strips of rat and rabbit bladder. Thus acetylcholine activates bladder smooth muscle via the m3 receptor subtype, and subsequent contractility may be transduced by guanine nucleotide binding proteins such as the Gi and Gq/11 subfamilies.     

Degeneration of neurons, synapses, and neuropil and glial activation in a murine Atm knockout model of ataxia-telangiectasia

Neural degeneration is one of the clinical manifestations of ataxia-telangiectasia, a disorder caused by mutations in the Atm protein kinase gene. However, neural degeneration was not detected with general purpose light microscopic methods in previous studies using several different lines of mice with disrupted Atm genes. Here, we show electron microscopic evidence of degeneration of several different types of neurons in the cerebellar cortex of 2-month-old Atm knockout mice, which is accompanied by glial activation, deterioration of neuropil structure, and both pre- and postsynaptic degeneration. These findings are similar to those in patients with ataxia-telangiectasia, indicating that Atm knockout mice are a useful model to elucidate the mechanisms underlying neurodegeneration in this condition and to develop and test strategies to palliate and prevent the disease.     

Pre- and post-synaptic muscarinic receptors in thin slices of rat adrenal gland

The effects of activation of muscarinic receptors on chromaffin cells and splanchnic nerve terminals were studied in a rat adrenal slice preparation. In chromaffin cells, muscarine induced a transient hyperpolarization followed by a depolarization associated with cell spiking. The hyperpolarization was blocked by charybdotoxin (1 microM) and tetraethylammonium chloride (TEA, 1 mM), but was not affected by 200 microM Cd2+ or removal of external Ca2+, consistent with activation of BK channels. This would follow internal Ca2+ mobilization, as shown by Ca2+ imaging with fura-2 on isolated chromaffin cells in culture. Under voltage-clamp, outward BK currents were insensitive to MT3 toxin, a specific muscarinic m4 receptor antagonist. In contrast, muscarine-induced depolarization was due to a m4 receptor-mediated inward current blocked by MT3 toxin. This current was permeable to cations and was associated with Ca2+ entry and subsequently, Ca2+-induced Ca2+ release. Finally, both muscarine (25 microM) and oxotremorine (10 microM) decreased the amplitude and frequency of KCI-evoked excitatory postsynaptic currents, without affecting quantal size, consistent with a presynaptic inhibitory effect. Taken together, our data suggest that activation of m4 and probably m3 muscarinic receptors results in a strong, long-lasting excitation of chromaffin cells, as well as an uncoupling of synaptic inputs onto these cells.     

Xanomeline: a novel muscarinic receptor agonist with functional selectivity for M1 receptors

Xanomeline [3(3-hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1- methylpyridine] has been evaluated as a muscarinic receptor agonist. In vitro, xanomeline had high affinity for muscarinic receptors in brain homogenates, but had substantially less or no affinity for a number of other neurotransmitter receptors and uptake sites. In cells stably expressing genetic m1 receptors, xanomeline increased phospholipid hydrolysis in CHO, BHK and A9 L cells to 100, 72 and 55% of the nonselective agonist carbachol. In isolated tissues, xanomeline had high affinity for M1 receptors in the rabbit vas deferens (IC50 = 0.006 nM), low affinity for M2 receptors in guinea pig atria (EC50 = 3 microM), was a weak partial agonist in guinea pig ileum and was neither an agonist nor antagonist in guinea pig bladder. In vivo, xanomeline increased striatal levels of dopamine metabolites, presumably by acting at M1 heteroreceptors on dopamine neurons to increase dopamine release. In contrast, xanomeline had only a relatively small effect on acetylcholine levels in brain, indicating that it is devoid of actions at muscarinic autoreceptors. In the gastrointestinal tract, xanomeline inhibited small intestinal and colonic motility, but increased small intestinal transmural potential difference. In contrast to the nonselective muscarinic agonist oxotremorine, xanomeline did not produce salivation, tremor nor hypothermia; it did, however, increase heart rate. The present data are consistent with the interpretation that xanomeline is a novel muscarinic receptor agonist with functional selectivity for M1 muscarinic receptors both in vitro and in vivo.     

Selectivity of LG50643 for postjunctional muscarinic-receptor subtype in the guinea-pig trachea

The effects of (+/-)-LG50643, a new N-quaternary tropinic ester of phenylcyclohexene carboxylic acid, endowed with a potent antimuscarinic activity, have been investigated on muscarinic receptor-mediated responses of the guinea-pig trachea to electrical field stimulation. An isolated preparation which allows the simultaneous measurement of tritiated acetylcholine release (prejunctional effect) and smooth muscle contraction (postjunctional effect) was used. The guinea-pig epithelium-deprived trachea was stimulated with 500 pulses (20 Hz, 1 ms, 9 V for 5 s, 30 s apart) in the presence of indomethacin (1 microM). Three successive pre- and postjunctional responses were observed. The potencies (-logEC50) of (+/-)-LG50643 for pre- and postjunctional muscarinic receptors were determined and compared with those of selective muscarinic antagonists. In addition, the affinity values of (+/-)-LG50643 for muscarinic-receptor subtypes were determined in radioligand binding experiments in cerebral cortex, heart and salivary glands of rat as target tissues for M1, M2 and M3 receptors, respectively. The results obtained in both functional and binding assays indicate (+/-)-LG50643 is a potent and selective antagonist for the M3-receptor subtype.     

Preexcitation secondary to fasciculoventricular pathways in children: a report of three cases

The clathrin-mediated sequestration pathway is used by non-G protein-coupled receptors (e.g., transferrin receptors) and a large number of G protein-coupled receptors, including beta-2 adrenoceptors and various muscarinic acetylcholine receptor (mAChR) subtypes. Recently, the ubiquitously expressed small GTPase RhoA has been implicated as a negative regulator of transferrin receptor internalization. Because mAChRs and other G protein-coupled receptors are able to activate RhoA, we investigated in HEK-293 cells whether RhoA regulates the sequestration of m1 and m2 mAChRs, which internalize via clathrin-coated and nonclathrin-coated vesicles in HEK-293 cells, respectively. Overexpression of wild-type RhoA inhibited agonist-induced sequestration of both m1 and m2 mAChRs by as much as 70%. Inhibition could be reversed by coexpression of Clostridium botulinum C3 transferase, which inactivates RhoA by ADP-ribosylation. Overexpression of C3 transferase alone had no effect on m1 and m2 mAChR sequestration. In addition, overexpression of RhoA inhibited m1 and m2 mAChR transport to the plasma membrane by 60 and 31%, respectively, which was blocked by coexpression of C3 transferase. We conclude that RhoA is not an endogenous regulator of mAChR sequestration, but when overexpressed, strongly inhibits mAChR trafficking (i.e., sequestration and transport to the plasma membrane) in HEK-293 cells.     

Pharmacological characterization of guanine nucleotide exchange reactions in membranes from CHO cells stably transfected with human muscarinic receptors m1-m4

We have studied muscarinic agonist stimulated [35S]GTP gamma S binding and [gamma 32P]GTP hydrolysis (GTPase) in membranes from CHO cells stably transfected with human muscarinic m1-m4 receptors. 'Full' agonists were at least 10-fold more potent at m2 & m4 receptors than at m1 & m3. This pattern was less marked with 'partial' agonists, which had a greater maximal effect at m2 & m4 than at m1 & m3. McN-A343 uniquely was more potent and efficacious at m4 than at m2 receptors. Antagonist affinity constants were estimated by fitting the data from inhibition curves directly to the Schild model. Antagonist affinity estimates were very similar to those measured earlier in binding studies using animal tissues, and confirmed a small degree of m4 selectivity for tropicamide and secoverine. The receptor subtypes activated more than one G-protein subtype; m2 & m4 receptors activated only pertussis (PTX) sensitive G-proteins, while m1 & m3 coupled to both PTX sensitive and insensitive G-proteins. Acetylcholine (ACh) was more potent in stimulating guanine nucleotide exchange in PTX-treated m1 cells than in controls.     

Arrestin-independent internalization of the m1, m3, and m4 subtypes of muscarinic cholinergic receptors

To understand what processes contribute to the agonist-induced internalization of subtypes of muscarinic acetylcholine receptors, we analyzed the role of arrestins. Whereas the m2 mAChR has been shown to undergo augmented internalization when arrestins 2 and 3 are overexpressed (Pals-Rylaarsdam, R., Gurevich, V. V., Lee, K. B., Ptasienski, J. A., Benovic, J. L., and Hosey, M. M. (1997) J. Biol. Chem. 272, 23682-23689), the agonist-induced internalization of m1, m3, and m4 mAChRs was unchanged when arrestins 2 or 3 were overexpressed in transiently transfected HEK-tsA201 cells. Furthermore, when a dominant-negative arrestin was used to interrupt endogenous arrestin function, there was no change in the internalization of the m1, m3, and m4 mAChR whereas the internalization of the beta2 adrenergic receptor was completely blocked. Wild-type and GTPase-deficient dominant-negative dynamin were used to determine which endocytic machinery played a role in the endocytosis of the subtypes of mAChRs. Interestingly, when dynamin function was blocked by overexpression of the GTPase-deficient dynamin, agonist- induced internalization of the the m1, m3, and m4 mAChRs was suppressed. These results suggested that the internalization of the m1, m3, and m4 mAChRs occurs via an arrestin-independent but dynamin-dependent pathway. To ascertain whether domains that confer arrestin sensitivity and dynamin insensitivity could be functionally exchanged between subtypes of mAChRs, chimeric m2/m3 receptors were analyzed for their properties of agonist-induced internalization. The results demonstrated that the third intracellular loop of the m2 mAChR conferred arrestin sensitivity and dynamin insensitivity to the arrestin-insensitive, dynamin-sensitive m3 mAChR while the analogous domain of the m3 mAChR conferred arrestin resistance and dynamin sensitivity to the previously arrestin-sensitive, dynamin-insensitive m2 mAChR.