Cardiac M2 Muscarinic Receptors Research Articles

The voltage-sensitive cardiac M2 muscarinic receptor modulates the inward rectification of the G protein-coupled, ACh-gated K+ current.

The acetylcholine (ACh)-gated inwardly rectifying K+ current (IKACh) plays a vital role in cardiac excitability by regulating heart rate variability and vulnerability to atrial arrhythmias. These crucial physiological contributions are determined principally by the inwardly rectifying nature of IKACh. Here, we investigated the relative contribution of two distinct mechanisms of IKACh inward rectification measured in atrial myocytes: a rapid component due to KACh channel block by intracellular Mg2+ and polyamines; and a time- and concentration-dependent mechanism. The time- and ACh concentration-dependent inward rectification component was eliminated when IKACh was activated by GTPγS, a compound that bypasses the muscarinic-2 receptor (M2R) and directly stimulates trimeric G proteins to open KACh channels. Moreover, the time-dependent component of IKACh inward rectification was also eliminated at ACh concentrations that saturate the receptor. These observations indicate that the time- and concentration-dependent rectification mechanism is an intrinsic property of the receptor, M2R; consistent with our previous work demonstrating that voltage-dependent conformational changes in the M2R alter the receptor affinity for ACh. Our analysis of the initial and time-dependent components of IKACh indicate that rapid Mg2+-polyamine block accounts for 60-70% of inward rectification, with M2R voltage sensitivity contributing 30-40% at sub-saturating ACh concentrations. Thus, while both inward rectification mechanisms are extrinsic to the KACh channel, to our knowledge, this is the first description of extrinsic inward rectification of ionic current attributable to an intrinsic voltage-sensitive property of a G protein-coupled receptor.

Increase in cardiac M2-muscarinic receptor expression is regulated by GATA binding protein 4 (GATA-4) in streptozotocin-induced diabetic rats

BackgroundAn increase in cardiac M2-muscarinic receptor (M2-mAChR) expression in diabetic rats has been observed, but the molecular mechanism of this increase remains unclear. The transcriptional activity of GATA binding protein 4 (GATA-4) has been documented to regulate the expression of M2-mAChR genes. In this study, we were interested in identifying the role of GATA-4 in the increase in M2-mAChR in diabetic rats and a primary culture of cardiomyocytes. MethodsStreptozotocin-induced diabetic rats (STZ-rats) and high-glucose (D-glucose 30mM, 24h)-treated primary cultures of cardiomyocytes from neonatal rats were used to investigate the role of GATA-4 in the change in M2-mAChR. The protein expression was determined by Western blot analysis. Phlorizin (Na+-glucose co-transport inhibitor), insulin, tiron (radical scavenger), PD98059 (ERK inhibitor) and SB203580 (p38 inhibitor) were used. We also silenced GATA-4 by RNAi to investigate the changes in M2-mAChR expression. ResultsThe cardiac output was reduced in STZ-rats with a higher expression of M2-mAChR or phosphorylated GATA-4 in the heart. These changes were reversed after correction of the blood sugar level. In cardiomyocytes, high glucose treatment also increased M2-mAChR expression and GATA-4 phosphorylation. These changes were reversed by tiron (ROS scavenger) or PD98059 (MEK/ERK inhibitor). However, an increase in M2-mAChR expression was not observed when GATA-4 was silenced by small interfering RNA (siRNA) in cardiomyocytes. ConclusionsWe suggest that hyperglycemia can cause a higher expression of M2-mAChR in cardiomyocytes mainly through ROS to enhance MEK/ERK for phosphorylation of GATA-4.

Increase of cardiac M2-muscarinic receptor gene expression in type-1 but not in type-2 diabetic rats

Changes of cardiac M2-muscarinic receptor (M2-mAChR) gene expression was investigated in type-1 like diabetic rats induced by intravenous injection of streptozotocin (STZ) and type-2 like diabetic rats induced by fed with fructose-rich chow. Systolic blood pressure (SBP) in STZ-diabetic rats was significantly lower than that in age-matched non-diabetic rats, while the SBP in type-2 like diabetic rats was higher than in non-diabetic rats. Also, the mRNA or protein level of cardiac M2-mAChR in STZ-diabetic rats was markedly higher than non-diabetic rats, but it was not observed in type-2 like diabetic rats as compared to age-matched non-diabetic rats. Arecaidine propargyl ester (APE), the agonist of M2-mAChR, produced a marked reduction of heart rate in STZ-diabetic rats but made less influence on heart rate in fructose-fed rats or non-diabetic rats. The results suggest that cardiac M2-mAChR gene expression is raised in type-1 like diabetic rats but not in type-2 like diabetic rats, this difference mainly due to hyperglycemia, for the production of hypotension in diabetic disorders.

Diethylphosphorylation of rat cardiac M2 muscarinic receptor by chlorpyrifos oxon in vitro

The acute toxicity of chlorpyrifos oxon (CPO), the metabolically-activated form of the major organophosphorus insecticide chlorpyrifos, is attributable to diethylphosphorylation of acetylcholinesterase at its esteratic site. As a secondary effect, CPO is known to compete with agonist binding to the M2 muscarinic acetylcholine receptor (mAChR). This study tests the hypothesis that [ethyl-1,2- 3H]CPO labels the M2 mAChR in rat cardiac membrane proteins. Of four labeled protein regions observed, only one had an apparent molecular mass (70–75 kDa) consistent with that of glycosylated M2 mAChR. It was identified as M2 muscarinic receptor by Western blotting and immunoprecipitation using a cardiac-specific M2 mAChR monoclonal antibody, providing the first direct evidence for diethylphosphorylation of a muscarinic receptor. This may be a functionally important M2 mAChR site, but the toxicological relevance and species and organ specificity of diethylphosphorylation are unknown.

Increased function of inhibitory neuronal M2 muscarinic receptors in diabetic rat lungs.

1. The function of inhibitory neuronal M2 muscarinic receptors in diabetic rat lungs was investigated. 2. Neuronal M2 muscarinic receptors inhibit acetylcholine release from parasympathetic nerves. Thus, stimulation of neuronal M2 muscarinic receptors with muscarinic agonists, such as pilocarpine, inhibits acetylcholine release and vagally induced bronchoconstriction. In contrast, blockade of neuronal M2 muscarinic receptors with selective M2 muscarinic antagonists, such as AF-DX 116, potentiates acetylcholine release and vagally induced bronchoconstriction. 3. Rats were made diabetic by streptozotocin (65 mg kg (-1), i.v.). After 7 14 days the rats were anaesthetized with urethane (1.5 g kg (-1), i.p.), tracheostomized, vagotomized, ventilated and paralysed with suxamethonium (30 mg kg (-1), i.v.). Some 7 day diabetic rats were treated with low doses of long acting (NPH) insulin (2 units day (-1), s.c.) for 7 days before experimentation. This dose of insulin was not sufficient to restore normoglycaemia in diabetic rats. Thus, insulin-treated diabetic rats remained hyperglycaemic. 4. Distal electrical stimulation (5 70 Hz, 6 s, 40 V, 0.4 ms) of the vagi caused bronchoconstriction, measured as an increase in inflation pressure and bradycardia. In diabetic rats, vagally induced bronchoconstriction was significantly depressed vs controls. In contrast, bronchoconstriction caused by i.v. acetylcholine was similar in diabetic and control animals. 5. The function of neuronal M2 muscarinic receptors was tested with the muscarinic agonist pilocarpine (0.001-100.0 microg kg (-1), i.v.) and the antagonist AF-DX 116 (0.01-3.0 mg kg (-1), i.v.). Pilocarpine inhibited vagally-induced bronchoconstriction (30 Hz, 20-40 V, 0.4 ms at 6 s) and AF-DX 116 potentiated vagally-induced bronchoconstriction (20 Hz, 20-40 V, 0.4 ms at 6 s) to a significantly greater degree in diabetic rats compared to controls. 6. Both frequency-dependent vagally-induced bronchoconstriction and M2 muscarinic receptor function could be restored to nearly control values in diabetic rats treated with low doses of insulin. 7. Displacement of [3H]QNB (1 nM) with the agonist carbachol (10.0 nM-10.0 mM) from diabetic cardiac M2 muscarinic receptors revealed a half log increase in agonist binding affinity at both the high and low affinity binding sites vs controls. In contrast, M2 receptors from insulin-treated diabetic rat hearts showed no significant difference in binding affinity vs controls. 8. These data show that neuronal M2 muscarinic receptors in the lungs have increased function in diabetic rats, suggesting that insulin modulates M2 muscarinic receptor function.

Studies on the interaction of steroidal neuromuscular blocking drugs with cardiac muscarinic receptors

Interaction of the steroidal neuromuscular blocking drugs pancuronium, pipecuronium, rocuronium and vecuronium with cardiac muscarinic receptors in rat hearts was investigated in vitro by a tritiated N-methyl hyoscine binding assay. We showed an interaction with cardiac muscarinic receptors with a rank order of potency pancuronium > vecuronium > pipecuronium > rocuronium and demonstrated complex binding characteristics for pancuronium, vecuronium and rocuronium, with "Hill coefficient" of less than unity. We conclude that the haemodynamic differences seen during the use of these neuromuscular blocking drugs may be a result of their interactions with cardiac M2 muscarinic receptors.