M2 Subtype Of Muscarinic Receptors Research Articles

Abstract 501: Selective i n vivo Modulation of Vagal-Muscarinic Signaling Prevents Sudden Cardiac Death in a Pressure Overload Model of Heart Failure

Introduction: Sudden cardiac death (SCD) from ventricular tachyarrhythmias (VT/VF) claims a quarter millions lives/year in USA. Patients with heart failure (HF) have high SCD risk. We previously demonstrated that differential remodeling of cardiac muscarinic receptors plays a critical role in pathogenesis and for effective therapy of HF and SCD risk [PMID: 25733594]. Ongoing clinical trials of chronic vagus nerve stimulation (cVNS) aimed at improving left ventricular (LV) function in HF patients have had mixed preliminary results. However, the effect of cVNS on SCD risk is unexplored. While acute VNS terminates VT/VF, cVNS likely alters the arrhythmogenic substrate. A better understanding of the underlying mechanisms is necessary for the design of new, more effective therapies. Hypothesis: cVNS prevents SCD by reducing calcium, autonomic and redox derangements in LV myocytes and improving repolarization reserve in pressure overloaded hearts. Methods: Guinea pigs are randomized to Sham or aortic constriction (AC) with daily brief (30-min) low-dose β-adrenergic stress (Shami; ACi), with and without in vivo cVNS therapy via an implanted device, with and without in vivo end-organ muscarinic receptor blockade (MRB) using glycopyrrolate. Echocardiography and continuous 24-hour ECG analyses are performed. Animals are sacrificed at 4 weeks for biochemical, excised perfused heart (pressure-volume, electrophysiology, optical mapping) and isolated LV myocyte studies at 37°C. PRELIMINARY RESULTS: Compared to Sham, the M2 subtype of muscarinic receptors are upregulated in AC and ACi, along with reduced calcium cycling, sarcomere shortening and β-adrenergic responsiveness. Whereas ACi+MRB markedly increased SCD incidence, ACi+cVNS prevented SCD. Interestingly, cVNS prolonged but also stabilized the ECG QT interval by reducing dispersion of repolarization in ACi model. Echocardiographic fractional shortening was also higher in ACi+cVNS (52±1%; 4 weeks) compared to ACi (27±2%; 4 weeks) and ACi+MRB (23±5%; 2 weeks). Conclusions: Chronic in vivo perturbation of cholinergic signaling may be an effective strategy for SCD therapy. Ongoing experiments are aimed at dissecting underlying mechanisms and identifying potential targets for SCD therapy.

Activation of M2 muscarinic receptors leads to sustained suppression of hippocampal transmission in the medial prefrontal cortex

Cholinergic innervation of the prefrontal cortex is critically involved in arousal, learning and memory. Dysfunction of muscarinic acetylcholine receptors and their downstream signalling pathways has been identified in mental retardation. To assess the role played by the muscarinic receptors at the hippocampal-frontal cortex synapses, an important relay in information storage, we used a newly developed frontal slice preparation in which hippocampal afferent fibres are preserved. Transient activation of muscarinic receptors by carbachol results in a long-lasting depression of synaptic efficacy at the hippocampal but not cortical pathways or local circuitry. On the basis of a combination of electrophysiological, pharmacological and anatomical results, this input-specific muscarinic modulation can be partially attributed to the M2 subtype of muscarinic receptors, possibly through a combination of pre- and postsynaptic mechanisms.

Cardiac Effect of Cholinesterase Inhibitors Used in Alzheimers Disease - from Basic Research to Bedside

In this review, the basic mechanism of the parasympathetic nervous effect on the heart is discussed. This is expanded to clinical situations to clarify what can happen to patients after cholinesterase (ChE) inhibitor is administered and to avoid unnecessary adverse effects. The parasympathetic nervous system can affect heart as well as brain function, and its effect on the heart is more complicated than is generally thought. The best-known effect is the cardioinhibitory effect, i.e. slowing of the heart rate. Its effect is also very sensitive to the time at which the stimulus falls within the cardiac cycle (phase-dependent effect). On some occasions, a cardiostimulatory effect can be observed. The parasympathetic nervous system also interacts with the sympathetic nervous system (sympathetic-parasympathetic interaction). ChE inhibitors or acetylcholinesterase inhibitors are often being administered to improve cognitive function of patients with Alzheimer's disease. The heart is naturally rich with ChE, and its inhibition may affect cardiac function, especially in elderly patients, many of whom have concomitant cardiovascular disease. Inhibition of ChE retards ACh degradation and potentiates the cardioinhibitory effect. However, the effect of ChE inhibitor is only slight in patients that receive a typical dose. After administration of ChE inhibitor in humans, the phase-dependent effect is reduced because the parasympathetic nervous effect is potentiated and saturated (saturation mechanism). Beat-by-beat fluctuation is reduced. ChE inhibitor increases arterial blood pressure through central M1 and M2 subtypes of muscarinic receptors (Br J Pharmacol 127:1657-1665, 1999). However, diastolic blood pressure increases slightly.

Potential synergy: voltage-driven steps in receptor-G protein coupling and beyond.

G protein-coupled receptor responses are not generally considered to show voltage dependence, but recently, the sensitivity of an oocyte to acetylcholine, which activates the m2 subtype of muscarinic receptors, was shown to be affected by changes in the membrane potential. Another example is the m2 receptor-evoked cation current in mammalian smooth muscle cells, which shows strong voltage dependence even when the receptor is bypassed by activating the G protein directly by intracellular application of GTPgammaS, a hydrolysis-resistant guanosine triphosphate (GTP) analog. Even among the few examples of voltage-dependent behavior known from among the plethora of G protein-coupled responses, the mechanism underlying the voltage dependence seems to differ.

Engagement of inducible nitric oxide synthase at the rostral ventrolateral medulla during mevinphos intoxication in the rat.

We evaluated the relationship between the toxicity induced by the organophosphate mevinphos (Mev) and inducible nitric oxide synthase (iNOS) in the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic neurogenic vasomotor tone. Adult Sprague-Dawley rats that were anesthetized and maintained with propofol were used. Laser scanning confocal microscopic analysis revealed colocalization of the M2 subtype of muscarinic receptors (M(2)R) and iNOS immunoreactivity in RVLM neurons. Comicroinjection bilaterally of Mev (10 nmol) and artificial cerebrospinal fluid (aCSF) into the RVLM elicited a progressive decline in systemic arterial pressure (SAP) and heart rate. This was accompanied during phase 1 Mev intoxication by an increase in the power density of the very high-frequency (VHF; 5-9 Hz), high-frequency (HF; 0.8-2.4 Hz), low-frequency (LF; 0.25- 0.8 Hz) and very low-frequency (VLF; 0-0.25 Hz) components of SAP signals. Phase 2 exhibited a reversal of the VHF and VLF power to control levels and a further reduction in the power density of both HF and LF components to below baseline. Hypotension and bradycardia promoted by Mev were significantly blunted on coadministration into the RVLM of the selective iNOS inhibitors S-methylisothiourea (250 pmol) or aminoguanidine (250 pmol). Not only was the augmented power density of HF and LF components during phase 1 Mev intoxication further enhanced, the reduced power of these two spectral components during phase 2 was appreciably antagonized. On the other hand, the temporal changes in VHF and VLF power were essentially the same as with coadministration of Mev and aCSF. We conclude that, as a cholinesterase inhibitor, Mev may induce toxicity via nitric oxide produced by iNOS on activation of the M(2)R by the accumulated acetylcholine in the RVLM.

Engagement of Inducible Nitric Oxide Synthase at the Rostral Ventrolateral Medulla during Mevinphos Intoxication in the Rat

We evaluated the relationship between the toxicity induced by the organophosphate mevinphos (Mev) and inducible nitric oxide synthase (iNOS) in the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic neurogenic vasomotor tone. Adult Sprague-Dawley rats that were anesthetized and maintained with propofol were used. Laser scanning confocal microscopic analysis revealed colocalization of the M2 subtype of muscarinic receptors (M₂R) and iNOS immunoreactivity in RVLM neurons. Comicroinjection bilaterally of Mev (10 nmol) and artificial cerebrospinal fluid (aCSF) into the RVLM elicited a progressive decline in systemic arterial pressure (SAP) and heart rate. This was accompanied during phase 1 Mev intoxication by an increase in the power density of the very high-frequency (VHF; 5–9 Hz), high-frequency (HF; 0.8–2.4 Hz), low-frequency (LF; 0.25– 0.8 Hz) and very low-frequency (VLF; 0–0.25 Hz) components of SAP signals. Phase 2 exhibited a reversal of the VHF and VLF power to control levels and a further reduction in the power density of both HF and LF components to below baseline. Hypotension and bradycardia promoted by Mev were significantly blunted on coadministration into the RVLM of the selective iNOS inhibitors S-methylisothiourea (250 pmol) or aminoguanidine (250 pmol). Not only was the augmented power density of HF and LF components during phase 1 Mev intoxication further enhanced, the reduced power of these two spectral components during phase 2 was appreciably antagonized. On the other hand, the temporal changes in VHF and VLF power were essentially the same as with coadministration of Mev and aCSF. We conclude that, as a cholinesterase inhibitor, Mev may induce toxicity via nitric oxide produced by iNOS on activation of the M₂R by the accumulated acetylcholine in the RVLM.

Daily variation of muscarinic receptors in visual cortex but not suprachiasmatic nucleus of Syrian hamsters

Intraventricular administration of carbachol can induce phase shifts in wheel-running activity in rodents, which depend on circadian phase and are mediated via muscarinic cholinergic receptors in Syrian hamsters. We studied the circadian variation in binding of [ 3 H ]- N-methylscopolamine ([ 3 H ]NMS), a hydrophilic muscarinic receptor antagonist, in micropunches obtained from the anterior hypothalamus and occipital cortex of Syrian hamsters housed in a 14:10 light:dark cycle. Binding sites were characterized on cells contained within 1 mm punches (obtained from slices 300 μm thick), using a method to selectively detect cell surface (functional) receptors. Atropine sulphate was used to determine nonspecific binding. Cortex showed a significant daily rhythm in [ 3 H ]NMS binding with a peak occurring late in the light phase and a trough at lights on, while the hypothalamus showed no detectable rhythm. Following suprachiasmatic nucleus (SCN) ablation or maintenance in constant darkness, the rhythm in the cortex was abolished. These findings suggest that photic information conveyed via the SCN is responsible for the receptor binding rhythm in the cortex. Autoradiographic studies ([ 3 H ]NMS; 2 nM, 3 weeks exposure) clearly revealed both M1 and M2 subtypes of muscarinic receptors in the region of the SCN and the visual cortex.

Methylene blue is a muscarinic antagonist in cardiac myocytes.

We studied the mechanism of action of methylene blue (Mblue), a putative guanylyl cyclase inhibitor, on the L-type calcium current (ICa) and the muscarinic activated K+ current (IK,ACh) in rat ventricular and atrial myocytes, respectively, and on the binding of [3H]quinuclidinyl benzylate in rat ventricular membranes. Superfusion, but not internal dialysis, with 30 microM Mblue antagonized the inhibitory effect of acetylcholine (ACh, 1 microM) on beta-adrenergic stimulation of ICa with isoprenaline (Iso, 10 nM or 1 microM). However, Mblue had no effect on the basal ICa or on the stimulation of ICa by Iso in the absence of ACh. The activation of IK,ACh by 3 microM ACh was also antagonized by Mblue in a dose-dependent manner. In contrast, Mblue had no effect on the activation of IK,ACh by either guanosine-5'-O-(3-thio)triphosphate or guanosine-5'-(beta,gamma-imido)triphosphate. Chlorpromazine (CPZ), a piperazine derivative like Mblue, also inhibited the muscarinic activation of IK,ACh in a dose-dependent manner. The specific binding of [3H]QNB, a muscarinic ligand, to rat ventricular membranes was displaced in a dose-dependent manner by Mblue and CPZ. The piperazine derivatives behaved like competitive antagonists of [3H]QNB binding, exhibiting equilibrium dissociation constant (Ki) values of 187 nM for Mblue and 366 nM for CPZ. In conclusion, Mblue exerts antimuscarinic effects on ICa and IK,ACh in rat cardiac myocytes that are best explained by the binding of Mblue to the M2 subtype of muscarinic receptors. This property probably contributes to the antimuscarinic effect of the putative guanylyl cyclase inhibitor reported in previous studies.

Acetylcholine-induced vasoconstrictor response of coronary vessels in rats: a possible contribution of M2 muscarinic receptor activation.

A mechanism by which acetylcholine (ACh) may elicit vasoconstrictor response in coronary vessels was studied in rat hearts perfused at a constant flow rate. In spontaneously beating hearts, bolus injections of ACh and carbachol (CCh) produced biphasic changes in coronary perfusion pressure (CPP): a transient increase at the initial period followed by a sustained decrease. In KCl-arrested hearts, ACh and CCh produced a monophasic increase in CPP, which was attenuated by either removal of endothelial cells by saponin or cyclooxygenase inhibition by diclofenac sodium. In the spontaneously beating heart, ACh-induced vasoconstriction was almost abolished by atropine (0.1 microM) and was markedly attenuated by an M2 antagonist, methoctramine (0.1 microM), but not by an M1 antagonist, pirenzepine (1 microM). Arecaidine propargyl ester (APE), an M2 agonist, produced coronary artery constriction which was attenuated by methoctramine (0.1 microM) but not by pirenzepine (0.1 microM) in both spontaneously beating and KCl-arrested hearts. McN-A-343, an M1 agonist, increased CPP in both beating and KCl-arrested hearts, but to a lesser degree than APE. These results suggest that the release of vasoconstrictor prostaglandins from endothelial cells contributes to the vasoconstrictor response to ACh in perfused rat coronary vessels, and the response to ACh appears to be mediated, in part, via the M2 subtype of muscarinic receptors.

Regulation of G protein-coupled receptor kinase activity

Recent progress on the activation of G protein-coupled receptor kinases is reviewed. beta-Adrenergic receptor kinase (beta ARK) is activated by G protein beta gamma -subunits, which interact with the carboxyl terminal portion of beta ARK. Muscarinic receptor m2-subtypes are phosphorylated by beta ARK1 in the central part of the third intracellular loop (I3). Phosphorylation of I3-GST fusion protein by beta ARK1 is synergistically stimulated by the beta gamma -subunits and mastoparan or a peptide corresponding to portions adjacent to the transmembrane segments of m2-receptors or by beta gamma -subunits and the agonist-bound I3-deleted m2 variant. These results indicate that agonist-bound receptors serve as both substrates and activators of beta ARK.

Correlation of the Anticholinesterase Activity of a Series of Organophosphates with Their Ability to Compete with Agonist Binding to Muscarinic Receptors

Some compounds that inhibit acetylcholinesterase (AChE) activity compete directly with quinuclidinyl benzilate (QNB) binding, a muscarinic antagonist which binds to all subtypes equally, and with cis-methyldioxolane (CD), an agonist that binds with high affinity to the M2 subtype of muscarinic receptors. The relationship between inhibition of AChE activity and the capability to affect muscarinic receptors directly has not been systematically explored. The interaction of eight organophosphates with muscarinic receptors was compared to their ability to inhibit AChE activity in vitro in tissue homogenates from rat hippocampus and frontal cortex, two cholinergically enriched areas of the brain. Of the compounds tested only echothiophate competed for [ 3H]QNB binding and only at concentrations greater than 100 μM. The anticholinesterase compounds were also tested for their ability to compete with a muscarinic receptor agonist, [ 3H]CD, which binds with high affinity (approximate K D = 3.5 nM) to 10 and 3% of the muscarinic receptors in the frontal cortex and hippocampus, respectively. The anticholinesterase compounds inhibited high-affinity [ 3H]CD binding up to 80% and the effects were similar in both tissues. Echothiophate and DFP were potent inhibitors of [ 3H]CD binding, as were the active "oxon" forms of parathion, malathion, and disulfoton. The parent "thio" forms of these insecticides, however, were much less effective in competing for [ 3H]CD binding. A similar pattern of potency was observed for the inhibition of brain AChE activity. A strong correlation was found between the ability of a compound to inhibit AChE activity and the ability to compete with [ 3H]CD binding. These data suggest that the biological effects of cholinesterase-inhibiting compounds may be due to more than their ability to inhibit AChE.

Cellular mapping of m2 muscarinic receptors in rat olfactory bulb using an antiserum raised against a cytoplasmic loop peptide

An antiserum that recognizes a sequence from the putative third cytoplasmic loop of the m2 subtype of muscarinic receptors (mAchR) has been raised and used to map the cellular distribution of this subtype in rat olfactory bulb. The antiserum was obtained by injecting BALB/C mice with a BSA-conjugated synthetic peptide whose sequence corresponded to amino acids 240–259 of the porcine cardiac m2 mAChR gene. Antibodies recognized the synthetic peptide in ELISA screening and labelled a single band corresponding to the peak of [ 3H]PrBCM-labelled heart mAchRs in immunoblots. Immunostaining of olfactory bulb, a region of the brain enriched in this muscarinic receptor subtype, showed that the antibodies labelled cell bodies and multiple dendritic processes. Broad fluorescent labelling throughout cell bodies was consistent with binding to the cytoplasmic face of the surface membrane, in support of the predicted cytoplasmic loop structure. m2-Positive cells throughout the bulb were sparsely distributed in different layers representing small subpopulations of the cells in each region: glomeruli, 6%; external plexiform layer, 16%; inner plexiform and granule cell layer, 3%. The results show that antibodies against specific sequences of different muscarinic receptor subtypes can be used to localize subtypes in situ, that the m2 subtype within the rat olfactory bulb is broadly distributed, and that the m2 subtype can occur postsynaptically in this central nervous system (CNS) region. The mapping of m2-positive cells in olfactory bulb may be of particular interest because loss of this subtype and degeneration of the olfactory system have been observed in Alzheimer's disease.

Differential allosteric effects of 8-( N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate-HCl (TMB-8) on muscarinic receptor subtypes

TMB-8, a putative inhibitor of intracellular calcium mobilization, prevents the binding of the muscarinic ligand N-[ 3H]methylscopolamine. The inhibition was observed in four tissues from guinea pig; cortex, heart, pancreas, and ileum, representing Ml, cardiac M2, glandular M2, and heterogeneous M2 subtypes of muscarinic receptors, respectively. The K i values for all four tissues were approx. 4 μM. However, dissociation kinetics revealed that TMB-8 interacted with an allosteric site of three muscarinic receptor subtypes but not the subtype from pancreas. These results indicate that TMB-8 interacts with muscarinic receptors, and therefore would disrupt calcium mobilization or any second messenger system coupled to these receptors.

Quantitative autoradiographic localization of the M1 and M2 subtypes of muscarinic receptors in monkey brain

Quantitative autoradiographic localization of the M1 and M2 subtypes of muscarinic receptors in monkey brain