Muscarinic receptor subtypes mediating central and peripheral antinociception studied with muscarinic receptor knockout mice: a review

To gain new insight into the physiological and pathophysiological roles of the muscarinic cholinergic system, we generated mutant mouse strains deficient in each of the five muscarinic acetylcholine receptor subtypes (M(1)-M(5)). In this chapter, we review a set of recent studies dealing with the identification of the muscarinic receptor subtypes mediating muscarinic agonist-dependent analgesic effects by central and peripheral mechanisms. Most of these studies were carried out with mutant mouse strains lacking M(2) or/and M(4) muscarinic receptors. It is well known that administration of centrally active muscarinic agonists induces pronounced analgesic effects. To identify the muscarinic receptors mediating this activity, wild-type and muscarinic receptor mutant mice were injected with the non-subtype-selective muscarinic agonist, oxotremorine (s.c., i.t., and i.c.v.), and analgesic effects were assessed in the tail-flick and hot-plate tests. These studies showed that M(2) receptors play a key role in mediating the analgesic effects of oxotremorine, both at the spinal and supraspinal level. However, studies with M(2)/M(4) receptor double KO mice indicated that M(4) receptors also contribute to this activity. Recent evidence suggests that activation of muscarinic receptors located in the skin can reduce the sensitivity of peripheral nociceptors. Electrophysiological and neurochemical studies with skin preparations from muscarinic receptor mutant mice indicated that muscarine-induced peripheral antinociception is mediated by M(2) receptors. Since acetylcholine is synthesized and released by different cell types of the skin, it is possible that non-neuronally released acetylcholine plays a role in modulating peripheral nociception. Our results highlight the usefulness of muscarinic receptor mutant mice to shed light on the functional roles of acetylcholine released from both neuronal and non-neuronal cells.     

Role of M1, M3, and M5 muscarinic acetylcholine receptors in cholinergic dilation of small arteries studied with gene-targeted mice

Acetylcholine regulates perfusion of numerous organs via changes in local blood flow involving muscarinic receptor-induced release of vasorelaxing agents from the endothelium. The purpose of the present study was to determine the role of M?, M?, and M? muscarinic acetylcholine receptors in vasodilation of small arteries using gene-targeted mice deficient in either of the three receptor subtypes (M1R(-/-), M3R(-/-), or M5R(-/-) mice, respectively). Muscarinic receptor gene expression was determined in murine cutaneous, skeletal muscle, and renal interlobar arteries using real-time PCR. Moreover, respective arteries from M1R(-/-), M3R(-/-), M5R(-/-), and wild-type mice were isolated, cannulated with micropipettes, and pressurized. Luminal diameter was measured using video microscopy. mRNA for all five muscarinic receptor subtypes was detected in all three vascular preparations from wild-type mice. However, M(3) receptor mRNA was found to be most abundant. Acetylcholine produced dose-dependent dilation in all three vascular preparations from M1R(-/-), M5R(-/-), and wild-type mice. In contrast, cholinergic dilation was virtually abolished in arteries from M3R(-/-) mice. Deletion of either M?, M?, or M? receptor genes did not affect responses to nonmuscarinic vasodilators, such as substance P and nitroprusside. These findings provide the first direct evidence that M? receptors mediate cholinergic vasodilation in cutaneous, skeletal muscle, and renal interlobar arteries. In contrast, neither M? nor M? receptors appear to be involved in cholinergic responses of the three vascular preparations tested.     

Investigations into the physiological role of muscarinic M2 and M4 muscarinic and M4 receptor subtypes using receptor knockout mice

Determination of muscarinic agonist-induced parasympathomimetic effects in wild type and M2 and M4 muscarinic receptor knockout mice revealed that M2 receptors mediated tremor and hypothermia, but not salivation. The M4 receptors seem to play a modest role in salivation, but did not alter hypothermia and tremor. In the M2 knockout mice, agonist-induced bradycardia in isolated spontaneously beating atria was completely absent compared to their wild type litter mates, whereas agonist-induced bradycardia was similar in the M4 knockout and wild type mice. The potency of carbachol to stimulate contraction of isolated stomach fundus, urinary bladder and trachea was reduced by a factor of about 2 in the M2 knockout mice, but was unaltered in the M4 knockout mice. The binding of the muscarinic agonist, [3H]-oxotremorine-M, was reduced in cortical tissue from the M2 knockout mice and to a lesser extent from the M4 knockout mice, and was reduced over 90% in the brain stem of M2 knockout mice. The data demonstrate the usefulness of knockout mice in determining the physiological function of peripheral and central muscarinic receptors.     

Alteration of cardiovascular and neuronal function in M1 knockout mice

We used gene targeting to generate mice lacking the M1 muscarinic acetylcholine receptor. These mice exhibit a decreased susceptibility to pilocarpine-induced seizures, loss of regulation of M-current potassium channel activity and of a specific calcium channel pathway in sympathetic neurons, a loss of the positive chronotropic and inotropic responses to the novel muscarinic agonist McN-A-343, and impaired learning in a hippocampal-dependent test of spatial memory.     

Novel insights into M5 muscarinic acetylcholine receptor function by the use of gene targeting technology

Until recently, little was known about the possible physiological functions of the M(5) muscarinic acetylcholine receptor subtype, the last member of the muscarinic receptor family (M(1)-M(5)) to be cloned. To learn more about the potential physiological roles of this receptor subtype, we generated and analyzed M(5) receptor-deficient mice (M5 -/- mice). Strikingly, acetylcholine, a potent dilator of most vascular beds, virtually lost the ability to dilate cerebral arteries and arterioles in M5 -/- mice, suggesting that endothelial M(5) receptors mediate this activity in wild-type mice. This effect was specific for cerebral blood vessels, since acetylcholine-mediated dilation of extra-cerebral arteries remained fully intact in M5 -/- mice. In addition, in vitro neurotransmitter release experiments indicated that M(5) receptors located on dopaminergic nerve terminals play a role in facilitating muscarinic agonist-induced dopamine release in the striatum, consistent with the observation that the dopaminergic neurons innervating the striatum almost exclusively express the M(5) receptor subtype. We also found that the rewarding effects of morphine, the prototypical opiate analgesic, were substantially reduced in M5 -/- mice, as measured in the conditioned place preference paradigm. Furthermore, both the somatic and affective components of naloxone-induced morphine withdrawal symptoms were significantly attenuated in M5 -/- mice. It is likely that these behavioral deficits are caused by the lack of mesolimbic M(5) receptors, activation of which is known to stimulate dopamine release in the nucleus accumbens. These results convincingly demonstrate that the M(5) muscarinic receptor is involved in modulating several important pharmacological and behavioral functions. These findings may lead to novel therapeutic strategies for the treatment of drug addiction and certain cerebrovascular disorders.     

Enhanced muscarinic M1 receptor gene expression in the corpus striatum of streptozotocin-induced diabetic rats

Acetylcholine (ACh), the first neurotransmitter to be identified, regulate the activities of central and peripheral functions through interactions with muscarinic receptors. Changes in muscarinic acetylcholine receptor (mAChR) have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). Previous reports from our laboratory on streptozotocin (STZ) induced diabetic rats showed down regulation of muscarinic M1 receptors in the brainstem, hypothalamus, cerebral cortex and pancreatic islets. In this study, we have investigated the changes of acetylcholine esterase (AChE) enzyme activity, total muscarinic and muscarinic M1 receptor binding and gene expression in the corpus striatum of STZ – diabetic rats and the insulin treated diabetic rats. The striatum, a neuronal nucleus intimately involved in motor behaviour, is one of the brain regions with the highest acetylcholine content. ACh has complex and clinically important actions in the striatum that are mediated predominantly by muscarinic receptors. We observed that insulin treatment brought back the decreased maximal velocity (V_max) of acetylcholine esterase in the corpus striatum during diabetes to near control state. In diabetic rats there was a decrease in maximal number (B_max) and affinity (K_d) of total muscarinic receptors whereas muscarinic M1 receptors were increased with decrease in affinity in diabetic rats. We observed that, in all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR experiment confirmed the increase in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. These results suggest the diabetes-induced changes of the cholinergic activity in the corpus striatum and the regulatory role of insulin on binding parameters and gene expression of total and muscarinic M1 receptors.     

Unrestrained nociceptive response and disregulation of hearing ability in mice lacking the nociceptin/orphaninFQ receptor.

In the G-protein-coupled receptor superfamily, the opioid receptor subfamily is constituted of the three distinct opioid receptors (namely delta-, mu- and kappa-subtypes) and the receptor for nociceptin (also designated orphaninFQ). The members of the opioid receptor subfamily were known to mediate a variety of cellular inhibitory effects. The three opioid receptors are known to play central roles in mediating analgesia and many other physiological activities; however, the nociceptin receptor was identified recently and less is known about its physiological roles. Here we report the generation and characterization of mice lacking the nociceptin receptor. The knockout mice showed no significant differences in nociceptive threshold and locomotor activity compared with control mice, but they lost nociceptin-induced behavioral responses. These results indicate that the nociceptin system is not essential for regulation of nociception or locomotor activity. On the other hand, we found insufficient recovery of hearing ability from the adaptation to sound exposure in the mutant mice. Thus, the nociceptin system appears to participate in the regulation of the auditory system.     

Sleep,activity, temperature and arousal responses of mice deficient for muscarinic receptor M2 or M4

AimsThe type 2 muscarinic receptor (M2R) differs from the other G-protein-coupled muscarinic receptor (type 4, or M4R) in tissue distribution and physiologic effects. We studied the impact of these receptors on sleep and arousal by using M2R and M4R knock-out (KO) mice.Main methodsM2R and M4R KO and genetically intact mice were compared in terms of normal patterns of sleep, responses to sleep loss, infectious challenge and acoustic startle, and acoustic prepulse inhibition of startle (PPI).Key findingsUnder basal conditions, M2R and M4R KO mice do not differ from the background strain or each other in the amount or diurnal pattern of sleep, locomotor activity, and body temperature. After enforced sleep loss, M2R KO mice, in contrast to the other two strains, show no rebound in slow-wave sleep (SWS) time, although their SWS is consolidated, and they show a greater rebound in time spent in REMS (rapid-eye-movement sleep) and REMS consolidation. During influenza infection, M2R KO mice, as compared with the other strains, show marked hypothermia and a less robust increase in SWS. During Candida albicans infection, M2R KO mice show a greater increase in SWS and a greater inflammatory response than do the other strains. M2R KO mice also show greater acoustic startle amplitude than does the background strain, although PPI was not different across the 3 strains over a range of stimulus intensities.SignificanceTaken together, these findings support different roles for M2R and M4R in the modulation of sleep and arousal during homeostatic challenge.     

Beneficial metabolic effects of M3 muscarinic acetylcholine receptor deficiency

Most animal models of obesity and hyperinsulinemia are associated with increased vagal cholinergic activity. The M3 muscarinic acetylcholine receptor subtype is widely expressed in the brain and peripheral tissues and plays a key role in mediating the physiological effects of vagal activation. Here, we tested the hypothesis that the absence of M3 receptors in mice might protect against various forms of experimentally or genetically induced obesity and obesity-associated metabolic deficits. In all cases, the lack of M3 receptors greatly ameliorated impairments in glucose homeostasis and insulin sensitivity but had less robust effects on overall adiposity. Under all experimental conditions tested, M3 receptor-deficient mice showed a significant elevation in basal and total energy expenditure, most likely due to enhanced central sympathetic outflow and increased rate of fatty-acid oxidation. These findings suggest that the M3 receptor may represent a potential pharmacologic target for the treatment of obesity and associated metabolic disorders.     

M5 muscarinic receptors are needed for slow activation of dopamine neurons and for rewarding brain stimulation

Mesopontine cholinergic neurons (Ch5 and Ch6 cell groups) activate the cerebral cortex via thalamic projections, and activate locomotion and reward via dopamine neurons in the substantia nigra and ventral tegmental area (VTA). Nicotinic receptors in VTA activate dopamine neurons quickly, and are needed for the stimulant and rewarding effects of nicotine in rats. Muscarinic receptors in VTA activate dopamine neurons slowly, and are needed for the rewarding effects of hypothalamic stimulation, but do not increase locomotion. Antisense oligonucleotides targetting M5 mRNA, when infused into the VTA, inhibited M5 receptor binding and rewarding hypothalamic stimulation. Mutant mice with truncated M5 muscarinic receptor genes drank more water than wild-type controls. Spontaneous locomotion and locomotor responses to amphetamine and scopolamine were unchanged. Electrical stimulation near Ch6 induced dopamine release in the nucleus accumbens in two phases, an early phase (0-2 min after stimulation) dependent on nicotinic and gluatamatergic receptors in VTA, and a late phase (8-50 min after stimulation) dependent on muscarinic receptors in VTA. The late phase was lost in M5 mutant mice, while the early phase was unchanged. M5 muscarinic receptors bind slowly to muscarinic ligands, and appear to mediate slow secretions.     

Muscarinic receptors: do they have a role in the pathology and treatment of schizophrenia?

The high affinity of antipsychotic drugs for the dopamine D2 receptor focused attention onto the role of these receptors in the genesis of psychoses and the pathology of schizophrenia. However, psychotic symptoms are only one aspect of the complex symptom profile associated with schizophrenia. Therefore, research continues into other neurochemical systems and their potential roles in key features associated with schizophrenia. Modulating the cholinergic system in attempts to treat schizophrenia predates specific neurochemical hypotheses of the disorder. Cholinergic modulation has progressed from the use of coma therapy, through the use of anti-cholinergic drugs to control side-effects of older (typical) antipsychotic medications, to the development of drugs designed to specifically activate selected muscarinic receptors. This review presents data implicating a decrease in muscarinic receptors, particularly the M1 receptor, in the pathology of schizophrenia and explores the potential physiological consequences of such a change, drawing on data available from muscarinic receptor knockout mice as well as clinical and pre-clinical pharmacology. The body of evidence presented suggests that deficits in muscarinic receptors are associated with some forms of schizophrenia and that targeting these receptors could prove to be of therapeutic benefit to patients with the disorder.     

Functional expression of M(1), M(3) and M(5) muscarinic acetylcholine receptors in yeast

The goal of this study was to functionally express the three G(q)-coupled muscarinic receptor subtypes, M(1), M(3) and M(5), in yeast (Saccharomyces cerevisiae). Transformation of yeast with expression constructs coding for the full-length receptors resulted in very low numbers of detectable muscarinic binding sites (B(max) < 5 fmol/mg). Strikingly, deletion of the central portion of the third intracellular loops of the M(1), M(3) and M(5) muscarinic receptors resulted in dramatic increases in B(max) values (53-214 fmol/mg). To monitor productive receptor/G-protein coupling, we used specifically engineered yeast strains that required agonist-stimulated receptor/G-protein coupling for cell growth. These studies showed that the shortened versions of the M(1), M(3) and M(5) receptors were unable to productively interact with the endogenous yeast G protein alpha-subunit, Gpa1p, or a Gpa1 mutant subunit that contained C-terminal mammalian Galpha(s) sequence. In contrast, all three receptors gained the ability to efficiently couple to a Gpa1/Galpha(q) hybrid subunit containing C-terminal mammalian Galpha(q) sequence, indicating that the M(1), M(3) and M(5) muscarinic receptors retained proper G-protein coupling selectivity in yeast. This is the first study to report the expression of muscarinic receptors in a coupling-competent form in yeast. The strategy described here, which involves structural modification of both receptors and co-expressed G proteins, should facilitate the functional expression of other classes of G protein-coupled receptors in yeast.     

Cloned M1 muscarinic receptors mediate both adenylate cyclase inhibition and phosphoinositide turnover

The rat M1 muscarinic receptor gene was cloned and expressed in a rat cell line lacking endogenous muscarinic receptors. Assignment of the cloned receptors to the M1 class was pharmacologically confirmed by their high affinity for the M1-selective muscarinic antagonist pirenzepine and low affinity for the M2-selective antagonist AF-DX-116. Guanylyl imidodiphosphate [Gpp(NH)p] converted agonist binding sites on the receptor, from high-affinity to the low-affinity state, thus indicating that the cloned receptors couple to endogenous G-proteins. The cloned receptors mediated both adenylate cyclase inhibition and phosphoinositide hydrolysis, but by different mechanisms. Pertussis toxin blocked the inhibition of adenylate cyclase (indicating coupling of the receptor to inhibitory G-protein), but did not affect phosphoinositide turnover. Furthermore, the stimulation of phosphoinositide hydrolysis was less efficient than the inhibition of adenylate cyclase. These findings demonstrate that cloned M1 receptors are capable of mediating multiple responses in the cell by coupling to different effectors, possibly to different G-proteins.     

G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation

G protein-coupled receptors (GPCRs) transduce extracellular signals into intracellular events. The waning responsiveness of GPCRs in the face of persistent agonist stimulation, or desensitization, is a necessary event that ensures physiological homeostasis. GPCR kinases (GRKs) are important regulators of GPCR desensitization. GRK5, one member of the GRK family, desensitizes central M(2) muscarinic receptors in mice. We questioned whether GRK5 might also be an important regulator of peripheral muscarinic receptor responsiveness in the cardiopulmonary system. Specifically, we wanted to determine the role of GRK5 in regulating muscarinic receptor-mediated control of airway smooth muscle tone or regulation of cholinergic-induced bradycardia. Tracheal pressure, heart rate, and tracheal smooth muscle tension were measured in mice having a targeted deletion of the GRK5 gene (GRK5(-/-)) and littermate wild-type (WT) control mice. Both in vivo and in vitro results showed that the airway contractile response to a muscarinic receptor agonist was not different between GRK5(-/-) and WT mice. However, the relaxation component of bilateral vagal stimulation and the airway smooth muscle relaxation resulting from beta(2)-adrenergic receptor activation were diminished in GRK5(-/-) mice. These data suggest that M(2) muscarinic receptor-mediated opposition of airway smooth muscle relaxation is regulated by GRK5 and is, therefore, excessive in GRK5(-/-) mice. In addition, this study shows that GRK5 regulates pulmonary responses in a tissue- and receptor-specific manner but does not regulate peripheral cardiac muscarinic receptors. GRK5 regulation of airway responses may have implications in obstructive airway diseases such as asthma or chronic obstructive pulmonary disease.     

M(3)-receptor knockout mice: muscarinic receptor function in atria, stomach fundus, urinary bladder, and trachea

Negative chronotropic and smooth muscle contractile responses to the nonselective muscarinic agonist carbamylcholine were compared in isolated tissues from M(3)-muscarinic receptor knockout and wild-type mice. Carbamylcholine (10(-8)-3.0 x 10(-5) M) induced a concentration-dependent decrease in atrial rate that was similar in atria from M(3)-receptor knockout and wild-type mice, indicating that M(3) receptors were not involved in muscarinic receptor-mediated atrial rate decreases. In contrast, the M(3) receptor was a major muscarinic receptor involved in smooth muscle contraction of stomach fundus, urinary bladder, and trachea, although differences existed in the extent of M(3)-receptor involvement among the tissues. Contraction to carbamylcholine was virtually abolished in urinary bladder from M(3)-receptor knockout mice, suggesting that contraction was predominantly due to M(3)-receptor activation. However, approximately 50-60% maximal contraction to carbamylcholine occurred in stomach fundus and trachea from M(3)-receptor knockout mice, indicating that contraction in these tissues was also due to M(2)-receptor activation. High concentrations of carbamylcholine relaxed the stomach fundus from M(3)-receptor knockout mice by M(1)-receptor activation. Thus M(3)-receptor knockout mice provided unambiguous evidence that M(3) receptors 1) play no role in carbamylcholine-induced atrial rate reduction, 2) are the predominant receptor mediating carbamylcholine-induced urinary bladder contractility, and 3) share contractile responsibility with M(2) receptors in mouse stomach fundus and trachea.     

Neuronal soma-dendritic and prejunctional M1-M4 receptors in gastrointestinal and genitourinary smooth muscle

A variety of neurons in gastrointestinal and genitourinary smooth muscle express muscarinic auto- as well as heteroreceptors. These receptors are found on the soma and dendrites of many cholinergic, sympathetic and NANC neurons and on axon terminals. A given neuron may contain both excitatory and inhibitory presynaptic muscarinic receptors. The subtypes involved are species- and tissue-dependent, and neuronal M1 to M4 receptors have been shown to be expressed in smooth muscle tissues. In this study, the ability of several selective muscarinic receptor antagonists to inhibit the effect of arecaidine propargyl ester (APE) on prejunctional muscarinic receptors on sympathetic nerve endings in the rabbit anococcygeus muscle (RAM) was investigated to characterise the receptor subtype involved. Electrical field stimulation (EFS) resulted in a release of noradrenaline (NA) eliciting monophasic contractions due to stimulation of postjunctional alpha1-adrenoceptors. The selective muscarinic agonist APE did not reduce contractions to exogenous NA, but caused a concentration-related and N-methylatropine-sensitive inhibition of neurogenic responses. All muscarinic antagonists investigated failed to affect the EFS-induced contractions, but shifted the concentration-response curve of APE to the right in a parallel and surmountable fashion. Schild analysis yielded regression lines of unit slope, indicating competitive antagonism. The following rank order of antagonist potencies (pA2 values) was found: tripitramine (9.10) > AQ-RA 741 (8.26) > or = himbacine (8.04) > or = (S)-dimethindene (7.69) > pirenzepine (6.46) > or = p-F-HHSiD (6.27). A comparison of the pA2 values determined in the present study with literature binding and functional affinities obtained at native or recombinant M1 to M5 receptors strongly suggests that NA release from sympathetic nerve endings in RAM is inhibited by activation of prejunctional muscarinic M2 receptors.     

M3 muscarinic receptor-deficient mice retain bethanechol-mediated intestinal ion transport and are more sensitive to colitis

Acetylcholine (ACh) is an important regulator of intestinal epithelial ion transport via muscarinic or nicotinic ACh receptors. Previous studies emphasize the role of the M3 muscarinic receptor subtype in mediating the effects of cholinergic agonists on intestinal ion transport. With the prevalence of mouse models to study intestinal (patho)physiology, it is crucial that ion transport be understood in this species. Using M3 receptor-deficient (KO) mice and wild-type (WT) mice, we examined M3 receptor contributions to ion transport as well as its role in colitis induced by dextran sodium sulphate (DSS). In the Ussing chambers, ileal and colonic tissue from M3 KO and WT mice displayed similar baseline ion transport properties. Short-circuit current (ISC) responses to the muscarinic receptor agonist bethanechol were slightly decreased in ileal tissue from M3 KO mice compared with tissue from WT mice, whereas responses were not significantly different in colonic tissue. ISC responses to bethanechol were partially inhibited by pirenzepine in WT ileum, but not tetrodotoxin, suggesting involvement of a non-neuronal M1 muscarinic receptor. In the ileum, the M3 receptor may inhibit neuronally evoked ion transport, as indicated by the increased ISC responses to electrical stimulation in tissue from M3 KO mice. Furthermore, whereas all DSS-treated mice developed colitis, M3 KO mice displayed more rapid mass loss and more severe disease than DSS-treated WT mice, even following a reduction in the amount and time of DSS treatment. Thus, M3 receptor-KO mice are compensated in their ability to evoke muscarinic receptor-driven ion transport responses, but are more sensitive to DSS. This work highlights the need to dissect muscarinic receptor-mediated events in the mouse, as mice become increasingly valuable in enteric disease models.     

Structural basis of M3 muscarinic receptor dimer/oligomer formation

Class A G protein-coupled receptors (GPCRs) are known to form dimers and/or oligomeric arrays in vitro and in vivo. These complexes are thought to play important roles in modulating class A GPCR function. Many studies suggest that residues located on the "outer" (lipid-facing) surface of the transmembrane (TM) receptor core are critically involved in the formation of class A receptor dimers (oligomers). However, no clear consensus has emerged regarding the identity of the TM helices or TM subsegments involved in this process. To shed light on this issue, we have used the M(3) muscarinic acetylcholine receptor (M3R), a prototypic class A GPCR, as a model system. Using a comprehensive and unbiased approach, we subjected all outward-facing residues (70 amino acids total) of the TM helical bundle (TM1-7) of the M3R to systematic alanine substitution mutagenesis. We then characterized the resulting mutant receptors in radioligand binding and functional studies and determined their ability to form dimers (oligomers) in bioluminescence resonance energy transfer saturation assays. We found that M3R/M3R interactions are not dependent on the presence of one specific structural motif but involve the outer surfaces of multiple TM subsegments (TM1-5 and -7) located within the central and endofacial portions of the TM receptor core. Moreover, we demonstrated that the outward-facing surfaces of most TM helices play critical roles in proper receptor folding and/or function. Guided by the bioluminescence resonance energy transfer data, molecular modeling studies suggested the existence of multiple dimeric/oligomeric M3R arrangements, which may exist in a dynamic equilibrium. Given the high structural homology found among all class A GPCRs, our results should be of considerable general relevance.     

Effect of M2 and M3 muscarinic receptors on airway responsiveness to carbachol in bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs.

The expression balance of M2 and M3 muscarinic receptor subtypes on the pathogenesis of airway hyperresponsiveness was investigated by using two congenitally related strains of guinea pigs, bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR). CCh-induced airway responses in vivo and in vitro were investigated by comparing the effects of muscarinic receptor subtype antagonists, and the relative amounts of M2 and M3 muscarinic receptor mRNA in tracheal smooth muscle and lung tissue were investigated. After treatment with muscarinic receptor subtype antagonists, the ventilatory mechanics (VT, Raw, and Cdyn) of response to CCh aerosol inhalation were measured by the bodyplethysmograph method. The effects of these antagonists on CCh-induced tracheal smooth muscle contraction were also investigated. The effects of M2 muscarinic receptor blockade were less but the effects of M3 muscarinic receptors blockade on the airway contractile responses were greater in BHS than in BHR. In M3 muscarinic receptor blockades, CCh-induced tracheal contractions in BHS were significantly greater than those in BHR. In tracheal smooth muscle from BHS, the relative amount of M2 muscarinic receptors mRNA was less but that of M3 muscarinic receptor mRNA was more than those in BHR. These results suggest that the high ACh level as a consequence of dysfunction of M2 muscarinic autoreceptors and the excessive effect of M3 muscarinic receptors on the airway smooth muscle may play an important role in the pathogenesis of airway hyperresponsiveness.     

A specific multi-nutrient formulation enhances M1 muscarinic acetylcholine receptor responses in vitro

Recent evidence indicates that supplementation with a specific combination of nutrients may affect cell membrane synthesis and composition. To investigate whether such nutrients may also modify the physical properties of membranes, and affect membrane-bound processes involved in signal transduction pathways, we studied the effects of nutrient supplementation on G protein-coupled receptor activation in vitro. In particular, we investigated muscarinic receptors, which are important for the progression of memory deterioration and pathology of Alzheimer's disease. Nerve growth factor differentiated pheochromocytoma cells that were supplemented with specific combinations of nutrients showed enhanced responses to muscarinic receptor agonists in a membrane potential assay. The largest effects were obtained with a combination of nutrients known as Fortasyn? Connect, comprising docosahexaenoic acid, eicosapentaenoic acid, uridine monophosphate as a uridine source, choline, vitamin B6, vitamin B12, folic acid, phospholipids, vitamin C, vitamin E, and selenium. In subsequent experiments, it was shown that the effects of supplementation could not be attributed to single nutrients. In addition, it was shown that the agonist-induced response and the supplement-induced enhancement of the response were blocked with the muscarinic receptor antagonists atropine, telenzepine, and AF-DX 384. In order to determine whether the effects of Fortasyn? Connect supplementation were receptor subtype specific, we investigated binding properties and activation of human muscarinic M1, M2 and M4 receptors in stably transfected Chinese hamster ovary cells after supplementation. Multi-nutrient supplementation did not change M1 receptor density in plasma membranes. However, M1 receptor-mediated G protein activation was significantly enhanced. In contrast, supplementation of M2- or M4-expressing cells did not affect receptor signaling. Taken together, these results indicate that a specific combination of nutrients acts synergistically in enhancing muscarinic M1 receptor responses, probably by facilitating receptor-mediated G protein activation.