M1 Subtype Of Muscarinic Receptor Research Articles

SUVN‐I6107: A True Positive Allosteric Modulator (PAM) at Muscarinic M1 Receptors for the Treatment of Cognitive Disorders

AbstractBackgroundMuscarinic acetylcholine receptors belong to the family of G‐protein coupled receptors and are expressed widely throughout the body. Muscarinic receptors have been implicated in the pathophysiology of various central nervous system disorders. M1 subtype of muscarinic receptors play a crucial role in cognition, attention, and sensory processing. The discovery and development of selective ligands targeting M1 subtype have been unsuccessful largely due to the highly conservative orthosteric binding site. Thus, ligands that selectively bind to a less conserved allosteric site of M1 receptors and positively modulate the orthosteric site may circumvent the selectivity issues as posed by orthosteric ligands.MethodSUVN‐I6107 is a clinical candidate identified from the M1 positive allosteric modulator (M1‐PAM) program. The effects of SUVN‐I6107 on the allosteric site of the M1 receptor were evaluated using the reporter gene assay. Binding liability towards orthosteric sites across all the muscarinic receptor subtypes was also evaluated. The pharmacokinetic properties of SUVN‐I6107 were studied both in rodent and non‐rodent species. The pro‐cognitive potential of SUVN‐I6107 was evaluated in diverse animal models of learning and memory. The safety of SUVN‐I6107 was assessed using various in vitro and in vivo systems.ResultSUVN‐I6107 was found to be a selective M1‐PAM with no agonistic activity at Muscarinic M1 and selective across other muscarinic receptor subtypes. SUVN‐I6107 showed good oral bioavailability in rat, dog and monkey. It also has excellent brain penetration with adequate protein free fraction. SUVN‐I6107 reversed the delay‐induced memory deficit in object recognition task. It also attenuated scopolamine‐induced memory deficit in the object recognition as well as contextual fear conditioning task. SUVN‐I6107 enhanced cerebral blood flow, potentiated the effects of donepezil and promoted non‐amyloidogenic amyloid precursor protein processing in rats. SUVN‐I6107 is devoid of side effects associated with the stimulation of the cholinergic system. SUVN‐I6107 was found to be safe in the safety pharmacology and phase‐1 enabling animal toxicity studies.ConclusionSUVN‐I6107, a novel, potent and selective M1‐PAM demonstrated efficacy in animal models and devoid of cholinergic side effects

Muscarinic Receptor–Dependent Long-Term Depression in Rat Visual Cortex Is PKC Independent but Requires ERK1/2 Activation and Protein Synthesis

Intact cholinergic innervation of visual cortex is critical for normal processing of visual information and for spatial memory acquisition and retention. However, a complete description of the mechanisms by which the cholinergic system modifies synaptic function in visual cortex is lacking. Previously it was shown that activation of the m1 subtype of muscarinic receptor induces an activity-dependent and partially N-methyl-d-aspartate receptor (NMDAR)-dependent long-term depression (LTD) at layer 4-layer 2/3 synapses in rat visual cortex slices in vitro. The cellular mechanisms downstream of the Galphaq coupled m1 receptor required for induction of this LTD (which we term mLTD) are currently unknown. Here, we confirm a role for m1 receptors in mLTD induction and use a series of pharmacological tools to study the signaling molecules downstream of m1 receptor activation in mLTD induction. We found that mLTD is prevented by inhibitors of L-type Ca(2+) channels, the Src kinase family, and the mitogen-activated kinase/extracellular kinase. mLTD is also partially dependent on phospholipase C but is unaffected by blocking protein kinase C. mLTD expression can be long-lasting (>2 h) and its long-term maintenance requires translation. Thus we report the signaling mechanisms underlying induction of an m1 receptor-dependent LTD in visual cortex and the requirement of protein synthesis for long-term expression. This plasticity could be a mechanism by which the cholinergic system modifies glutamatergic synapse function to permit normal visual system processing required for cognition.

Acetylcholine and Acetylcholine Receptors in Taste Receptor Cells

Neuroactive substances play important roles as transmitters and neuromodulators. Although many of these substances and/or their receptors are known to be present in taste receptor cells and nerve fibers innervating taste buds, physiological functions of these substances are not well understood (Nagai et al., 1996). Using Ca2+ imaging and immunocytochemical techniques, we have examined the physiological responses of taste receptor cells to acetylcholine (ACh), classified the types of ACh receptors, and determined the underlying signaling mechanisms in taste receptor cells. Our results suggest that ACh may be involved in cell-to-cell communication within the taste bud and in neuromodulation of taste transduction mechanisms. In Ca 2+ -imaging study, freshly isolated taste receptor cells were loaded with Ca2+-sensitive fluorescent dye Fura-2 and intracellular Ca2+ levels were measured ratiometrically. ACh induced increases in intracellular Ca 2+ levels ([Ca 2+ ]i) in taste receptor cells of mouse, rat and mudpuppy. The magnitude of the peak Ca 2+ response to ACh was concentration-dependent with half-maximum responses around 1 µM. To determine which subtypes of ACh receptors and signaling pathway were involved, we examined the effect of receptor antagonists and inhibitors for selective pathway. Atropine (0.5 µM), a muscarinic ACh receptor antagonist, blocked the ACh response, while D-tubocurarine (250 µM), a nicotinic ACh receptor antagonist, had no effect. In addition, the phospholipase C (PLC) inhibitor U73122 (5 µM) and the Ca 2+ -ATPase inhibitor thapsigargin (1 µM), which depletes intracellular Ca 2+ stores, blocked the ACh responses. These results suggest that ACh binds to muscarinic ACh receptors, which activates PLC, resulting in the production of IP3 and the subsequent release of Ca 2+ from the IP3-sensitive-intracellular stores. Since it is known that binding of ACh to muscarinic receptor subtypes M1/M3/M5 activates the PLC signaling pathways (Caulfield, 1993), our data indicates the presence of at least one of these receptors in taste receptor cells. Additionally, we found that prolonged stimulation (>1 min) with ACh (10 µM) induced a biphasic response with a transient followed by a sustained [Ca 2+ ]i increase. The sustained phase of the [Ca 2+ ]i increase was dependent on Ca 2+ influx as removal of extracellular Ca 2+ eliminated the response. Subsequently adding external Ca 2+ induced increases in [Ca2+] i , suggesting Ca2+ entry through Ca2+permeable channels. This is consistent with our previous studies showing presence of Ca 2+ store-operated channels (SOC) in taste cells (Ogura, 2002; Ogura et al., 2002). SOCs are activated solely by store depletion without requirement of a receptor-mediated mechanism, a mechanism also known as ‘capacitative calcium entry’. Thus it is possible that the sustained part of the ACh-induced calcium response is mediated by Ca 2+ influx through SOCs. In immunocytochemical study, sections containing rat circumvallate and foliate papillae were immunoreacted with an antiserum against the M1 subtype of muscarinic ACh receptors. Positive reaction was observed in many taste cells of each taste bud. In crosssections of rat circumvallate papillae, roughly half of the taste cells were immunolabeled. No selective labeling was observed in control sections, in which primary antibody was omitted. Preabsorption with antigen significantly reduced the labeling. This result suggests that taste receptor cells express M1 subtype of ACh receptor. Thus ACh can bind to the M1 subtype of the muscarinic receptors and activate the PLC/IP3 pathway. To study whether ACh is stored in synaptic vesicles in taste receptor cells and/or adjacent nerve fibers, we immunolabeled the vesicular ACh transporter (VAChT), a key element of AChcontaining vesicle in mouse taste tissue. A subset of taste receptor cells exhibited positive immunoreactivity to the antibody against VAChT. In addition, certain nerve fibers surrounding or within taste buds are positively reacted to antibodies against VAChT. These results suggest that taste receptor cells could release ACh for cell-tocell communications among taste receptor cells and/or synaptic transmission from taste receptor cells to taste sensory fibers. The presence of VAChT in adjacent nerve fibers also reveals a possibility of cholinergic modulations of taste receptor cells via the muscarinic receptors. Taken together, our results demonstrate ACh responses and its signaling pathway in taste receptor cells. Since ACh increases [Ca 2+ ]i via PLC-mediated pathway, ACh may regulate taste responses by means of changing [Ca 2+ ]i levels or PLC signaling. It is known that

Acetylcholine increases intracellular Ca2+ in taste cells via activation of muscarinic receptors.

Previous studies suggest that acetylcholine (ACh) is a transmitter released from taste cells as well as a transmitter in cholinergic efferent neurons innervating taste buds. However, the physiological effects on taste cells have not been established. I examined effects of ACh on taste-receptor cells by monitoring [Ca2+]i. ACh increased [Ca2+]i in both rat and mudpuppy taste cells. Atropine blocked the ACh response, but D-tubocurarine did not. U73122, a phospholipase C inhibitor, and thapsigargin, a Ca2+-ATPase inhibitor that depletes intracellular Ca2+ stores, blocked the ACh response. These results suggest that ACh binds to M1/M3/M5-like subtypes of muscarinic ACh receptors, causing an increase in inositol 1,4,5-trisphosphate and subsequent release of Ca2+ from the intracellular stores. A long incubation with ACh induced a transient response followed by a sustained phase of [Ca2+]i increase. In Ca2+-free solution, the sustained phases disappeared, suggesting that Ca2+ influx is involved in the sustained phase. Depletion of Ca2+ stores by thapsigargin alone induced Ca2+ influx. These findings suggest that Ca2+ store-operated channels may be present in taste cells and that they may participate in the sustained phase of [Ca2+]i increase. Immunocytochemical experiments indicated that the M1 subtype of muscarinic receptors is present in both rat and mudpuppy taste cells.

Carbachol and acetylcholine delay the early postdenervation depolarization of muscle fibres through M1-cholinergic receptors.

The resting membrane potential (RMP) of denervated muscle fibres of rat diaphragm muscle is depolarized by approximately 8-10 mV during the first 3 h after nerve section and this early postdenervation depolarization is reduced substantially by the presence of 5x10(-8) M acetylcholine (ACh) or carbachol (CB). The muscarinic antagonist atropine (Atr; 5x10(-9) to 5x10(-6) M) reduced the effect of CB in a dose-dependent manner (K(i)=7x10(-8) M) and increased the rate of the early postdenervation depolarization. In lower doses (5x10(-7) M), Atr acted only in the presence of an allosteric stabilizator hexamethylene-bis-[dimethyl-(3-phtalimidopropyl)ammonium] (W-84). Also pirenzepine, a specific inhibitor of the M1 subtype of muscarinic receptor, blocked the action of CB in a dose-dependent manner with an apparent inhibition constant K(i)=1x10(-7) microM. DAMP, a specific M3 antagonist, was without effect on the muscle hyperpolarization induced by CB. CB also hyperpolarized the membrane potentials of muscles which were denervated for 1-3 days. It is concluded that ACh and CB protect the muscle fibres from early depolarization through M1-cholinergic receptors on the muscle membrane. These particular receptors can apparently mediate the 'trophic', non-impulse regulation of RMP in skeletal muscles when they are activated by acetylcholine released non-quantally.

Activation of nitric oxide signaling through muscarinic receptors in submandibular glands by primary Sjögren syndrome antibodies.

Primary Sjögren Syndrome is a chronic autoimmune disease characterized by exocrine gland dysfunction. Here we present evidence of the activation of nitric oxide signaling cascade by circulating antibodies of patients with Sjögren Syndrome in rat submandibular glands. Constitutive nitric oxide synthase and cyclic GMP levels are modulated by Sjögren IgGs through the activation of muscarinic acetylcholine receptors on the glands. The effects are similar to those produced by the agonist carbachol and blocked by the antagonist atropine. The involvement of M1 subtype of muscarinic receptors is proposed since both a synthetic peptide homologous to an extracellular domain of M1 receptor and pirenzepine, a selective M1 antagonist, partially blocked the effects. We conclude that Sjögren Syndrome antibodies can activate nitric oxide signaling in submandibular glands by interacting with muscarinic acetylcholine receptors.

Selective allosteric enhancement of the binding and actions of acetylcholine at muscarinic receptor subtypes.

The ternary allosteric model predicts the possibility of discovering molecules with novel and highly subtype-selective modes of action. This approach has been applied to muscarinic receptors. The alkaloid brucine is capable of selectively enhancing by an allosteric mechanism the effects of low but not high concentrations of acetylcholine at only the m1 subtype of muscarinic receptors. A simple derivative of brucine, N-chloromethylbrucine, enhances acetylcholine actions selectively at only m3 receptors. In addition it binds to, but does not affect, the properties of m4 receptors, thereby demonstrating neutral cooperativity and an 'absolute' selectivity of action at m3 receptors over m4 receptors. Brucine N-oxide enhances acetylcholine binding at m3 and m4 receptors and is neutral at m1 and m5 receptors. These findings allow the possibility of developing muscarinic agents that have a novel and highly targeted mode of action; they may act only on a single muscarinic receptor subtype which is functioning sub-optimally and therefore be of use therapeutically in the early stages of Alzheimer's Disease.

Muscarinic receptor-dependent activation of phospholipase C in human fetal central nervous system organotypic tissue culture

The coupling of muscarinic-cholinergic receptors (mAchR) with the phospholipase C (PLC) second messenger system has been demonstrated in central nervous system (CNS) tissue of many animal species. However, little information exists regarding this association in the developing human CNS. Due to the suggested role of acetylcholine in the regulation of development and differentiation of neural cells, the knowledge of these relationships during human fetal development acquires singular importance. Because of this, we examined the cholinergic stimulation of PLC in human fetal CNS organotypic tissue cultures. Agonist treatment of cultures, in the presence of lithium, resulted in a 4–6-fold increase in inositol phosphates formation. This increase was caused principally by the formation of inositol phosphate (IP). However, kinetic studies demonstrated that the levels of IP 2, IP 3 and IP 4 also increased rapidly after stimulation reaching maximum levels before IT. These results support the hypothesis that muscarinic receptor activation results in an increase in the hydrolysis of PIP 2. The inositol phosphate formation was dependent on agonist concentration. The obtained EC 50 values were approximately 57 ± 15 μM for carbachol, 8 ± 2 μM for acetylcholine and 49 ± 15 μM for oxotremorine. The agonist-dependent formation of inositol phosphates was inhibited by the muscarinic antagonists atropine and pirenzepine. Pirenzepine inhibited carbachol stimulation with high affinity ( K i = 2.90 ± 1.15 nM), indicating that PLC activation is the result of activation of the m1 subtype of muscarinic receptors. Treatment of cultures with pertussis toxin did not result in inhibition of agonist-dependent activation of PLC. This result suggests that the m1 muscarinic receptor is coupled to PLC through G q.

Muscarinic receptor M1 subtypes coupling to inositol phospholipid metabolism in cerebral cortex of rats following a repeated treatment with diisopropyl fluorophosphate

Muscarinic receptor M1 subtypes coupling to inositol phospholipid metabolism in cerebral cortex of rats following a repeated treatment with diisopropyl fluorophosphate

A muscarinic receptor subtype modulates vagally stimulated bronchial contraction.

An in vitro preparation was developed to study vagus nerve-stimulated (preganglionic) and field-stimulated (post-ganglionic) contraction of the rabbit main stem bronchus and to compare the inhibitory effects of muscarinic antagonists on that contraction. The maximal contractile responses (20 V, 0.5 ms, 64 Hz) for either field or vagal stimulation were completely abolished by atropine (60 nM). Hexamethonium (0.1 mM) abolished the response to vagal stimulation but did not affect the field-stimulated response. To compare the effectiveness of atropine and pirenzepine as antagonists at the nerve-smooth muscle junction, inhibition studies of field-stimulated contractions were performed. Pirenzepine was 102- to 178-fold less potent than atropine when compared at the inhibitory concentration of antagonist that produced 25, 50, and 75% inhibition (IC25, IC50, and IC75, respectively), indicating that the muscarinic receptor at the nerve-smooth muscle junction is a muscarinic receptor with low affinity for pirenzepine (M2 subtype). Atropine had similar inhibitory effects on vagal- and field-stimulated contractions. In contrast, pirenzepine was more potent in inhibiting vagally stimulated contraction than field-stimulated contraction, especially at the IC25 where pirenzepine was only 8- to 22-fold less potent than atropine in inhibiting vagally stimulated contraction. These data suggest that an M1 subtype of muscarinic receptor modulates excitatory neurotransmission through bronchial parasympathetic ganglia.

Vagal cholinergic control of gastric alkaline secretion in normal subjects and duodenal ulcer patients.

Gastric alkaline secretion was determined in ranitidine treated healthy subjects and duodenal ulcer (DU) patients using gastric perfusion aspiration system and back titration of gastric perfusate to original pH 6.0. Basal alkaline secretion showed periodic fluctuations reaching peaks at phase III of the migrating motor complex (MMC) in the stomach. Mean basal alkaline secretion in healthy normals and DU patients averaged 1120 +/- 124 and 880 +/- 72 mumol/h, respectively and no correlation was found between basal and maximally stimulated gastric acid and alkaline secretion. Modified sham feeding in normal subjects and in DU patients increased this secretion to the peaks of about 28 and 36% of the maximal alkaline response to intragastric application of 16,16 dimethyl-PGE2 in these subjects. Vagotomy did not affect significantly basal alkaline secretion but prevented the rise in alkaline secretion induced by modified sham feeding. Atropine (5-20 micrograms/kg) decreased dose dependently basal, and prevented the modified sham feeding induced alkaline secretion, while pirenzepine (5-20 micrograms/kg) had little influence on basal, and did not affect the modified sham feeding induced, alkaline secretion. This study shows that basal gastric alkaline secretion fluctuates in phase with gastric motor activity, and is similar in normal and DU patients. Vagal stimulation strongly increases alkaline secretion, the effect being abolished by vagotomy and atropine, but not by pirenzepine, suggesting the involvement of M2 rather than M1 subtypes of muscarinic receptors in this stimulation.

Muscarinic ml receptors in the lateral septal area mediate cardiovascular responses to cholinergic agonists and bradykinin: supersensitivity induced by chronic treatment with atropine

The infusion of pilocarpine, acetylcholine, bradykinin and the selective Ml muscarinic agonist McNeil-A-343 into the lateral septal area produced a dose-dependent increase of arterial blood pressure and heart rate. The M2 muscarinic agonist carbamylcholine that causes a rise in arterial blood pressure when injected into the anterior lateral ventricles did not produce any cardiovascular effects when infused into the lateral septal area. Chronic treatment with atropine induced supersensitivity to the muscarinic agonists and a significant increase in the number of muscarinic receptors. In this study bradykinin failed to produce any significant change in cardiovascular activity. Pirenzepine, a M1 muscarinic blocking agent, inhibited completely the effect of both muscarinic agonists and bradykinin on cardiovascular activity. In fact, in vitro studies shows that the displacement of the binding of [ 3H]QNB by pirenzepine is compatible with the presence of the M1 subtype of muscarinic receptor in the lateral septal area, where it may play a major role on cardiovascular regulation.

Identification of muscarinic receptor subtype mediating colonic response to eating

The colonic motor response to eating requires cholinergic transmission. Recent studies have identified two subclasses of muscarinic receptor, the M1 and the M2 subtype. The aim of this study was to evaluate the muscarinic receptor subtype responsible for mediating the gastrocolonic response. Spike potential (SP) activity and intraluminal pressure were recorded, during fasting and after eating a 1000-kcal mixed meal, from the distal colon of 10 healthy volunteers. In each subject three paired studies were carried out: either atropine (a nonselective antimuscarinic, 1 mg), pirenzepine (a selective M1 antimuscarinic, 10 mg), or saline were infused intravenously before eating, using a double-blind crossover design. The meal significantly increased colonic spike potential activity (17.6 +/- 3.9, SP/30 min) above fasting values (2.7 +/- 0.6, P less than 0.01) in the control study. There was no postprandial increase in spike potential activity (3.3 +/- 0.9, SP/30 min) after atropine. The meal also significantly increased spike potential activity (15.4 +/- 3.3 SP/30 min, P less than 0.01) above fasting levels and equal to that of the controls, in the pirenzepine study. These data suggest that the colonic motor response to eating is mediated through the M2 but not the M1 subtype of muscarinic receptors.