A cellular mechanism for nitric oxide-mediated cholinergic control of mammalian heart rate

The biochemical signaling pathways involved in nitric oxide (NO)- mediated cholinergic inhibition of L-type Ca2+ current (ICa[L]) were investigated in isolated primary pacemaker cells from the rabbit sinoatrial node (SAN) using the nystatin-perforated whole-cell voltage clamp technique. Carbamylcholine (CCh; 1 microM), a stable analogue of acetylcholine, significantly inhibited ICa(L) after it had been augmented by isoproterenol (ISO; 1 microM). CCh also activated an outward K+ current, IK(ACh). Both of these effects of CCh were blocked completely by atropine. Preincubation of the SAN cells with L-nitro- arginine methyl ester (L-NAME; 0.2-1 mM), which inhibits NO synthase (NOS), abolished the CCh-induced attenuation of ICa(L) but had no effect on IK(ACh). Coincubation of cells with both L-NAME and the endogenous substrate of NOS, L-arginine (1 nM), restored the CCh- induced attenuation of ICa(L), indicating that L-NAME did not directly interfere with the muscarinic action of CCh on ICa(L). In the presence of ISO the CCh-induced inhibition of ICa(L) could be mimicked by the NO donor 3-morpholino-sydnonimine (SIN-1; 0.1 mM). SIN-1 had no effect on its own or after a maximal effect of CCh had developed, indicating that it does not inhibit ICa(L) directly. SIN-1 failed to activate IK(ACh), demonstrating that it did not activate muscarinic receptors. Both CCh and NO are known to activate guanylyl cyclase and elevate intracellular cGMP. External application of methylene blue (10 microM), which interferes with the ability of NO to activate guanylyl cyclase, blocked the CCh-induced attenuation of ICa(L). However, it also blocked the activation of IK(ACh), suggesting an additional effect on muscarinic receptors or G proteins. To address this, a separate series of experiments was performed using conventional whole-cell recordings with methylene blue in the pipette. Under these conditions, the CCh-induced attenuation of ICa(L) was blocked, but the activation of IK(ACh) was still observed. Methylene blue also blocked the SIN-1-induced decrease in ICa(L). 6-anilino-5,8-quinolinedione (LY83583; 30 microM), an agent known to decrease both basal and CCh-stimulated cGMP levels, prevented the inhibitory effects of both CCh and SIN-1 on ICa(L), but had no effect on the activation of IK(ACh) by CCh. In combination, these results show that CCh- and NO-induced inhibition of ICa(L) is mediated by cGMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Novel inhibition of gbetagamma-activated potassium currents induced by M(2) muscarinic receptors via a pertussis toxin-insensitive pathway

G(i) protein-coupled receptors such as the M(2) muscarinic acetylcholine receptor (mAChR) and A(1) adenosine receptor have been shown to activate G protein-activated inwardly rectifying K(+) channels (GIRKs) via pertussis toxin-sensitive G proteins in atrial myocytes and in many neuronal cells. Here we show that muscarinic M(2) receptors not only activate but also reversibly inhibit these K(+) currents when stimulated with agonist for up to 2 min. The M(2) mAChR-mediated inhibition of the channel was also observed when the channels were first activated by inclusion of guanosine 5'-O-(thiotriphosphate) in the pipette. Under these conditions the M(2) mAChR-induced inhibition was quasi-irreversible, suggesting a role for G proteins in the inhibitory process. In contrast, when GIRK currents were maximally activated by co-expressing exogenous Gbetagamma, the extent of acetylcholine (ACh)-induced inhibition was significantly reduced, suggesting competition between the receptor-mediated inhibition and the large pool of available Gbetagamma subunits. The signaling pathway that led to the ACh-induced inhibition of GIRK channels was unaffected by pertussis toxin pretreatment. Furthermore, the internalization and agonist-induced phosphorylation of M(2) mAChR was not required because a phosphorylation- and internalization-deficient mutant of the M(2) mAChR was as potent as the wild-type counterpart. Pharmacological agents modulating various protein kinases or phosphatidylinositol 3-kinase did not affect the inhibition of GIRK currents. Furthermore, the signaling pathway that mediates GIRK current inhibition was found to be membrane-delimited because bath application of ACh did not inhibit GIRK channel activity in cell-attached patches. Other G protein-coupled receptors including M(4) mAChR and alpha(1A) adrenergic receptors also caused the inhibition, whereas other G protein-coupled receptors including A(1) and A(3) adenosine receptors and alpha(2A) and alpha(2C) adrenergic receptors could not induce the inhibition. The presented results suggest the existence of a novel signaling pathway that can be activated selectively by M(2) and M(4) mAChR but not by adenosine receptors and that involves non-pertussis toxin-sensitive G proteins leading to an inhibition of Gbetagamma-activated GIRK currents in a membrane-delimited fashion.     

Human tenocytes are stimulated to proliferate by acetylcholine through an EGFR signalling pathway

Studies of human patellar and Achilles tendons have shown that primary tendon fibroblasts (tenocytes) not only have the capacity to produce acetylcholine (ACh) but also express muscarinic ACh receptors (mAChRs) through which ACh can exert its effects. In patients with tendinopathy (chronic tendon pain) with tendinosis, the tendon tissue is characterised by hypercellularity and angiogenesis, both of which might be influenced by ACh. In this study, we have tested the hypothesis that ACh increases the proliferation rate of tenocytes through mAChR stimulation and have examined whether this mechanism operates via the extracellular activation of the epidermal growth factor receptor (EGFR), as shown in other fibroblastic cells. By use of primary human tendon cell cultures, we identified cells expressing vimentin, tenomodulin and scleraxis and found that these cells also contained enzymes related to ACh synthesis and release (choline acetyltransferase and vesicular acetylcholine transporter). The cells furthermore expressed mAChRs of several subtypes. Exogenously administered ACh stimulated proliferation and increased the viability of tenocytes in vitro. When the cells were exposed to atropine (an mAChR antagonist) or the EGFR inhibitor AG1478, the proliferative effect of ACh decreased. Western blot revealed increased phosphorylation, after ACh stimulation, for both EGFR and the extracellular-signal-regulated kinases 1 and 2. Given that tenocytes have been shown to produce ACh and express mAChRs, this study provides evidence of a possible autocrine loop that might contribute to the hypercellularity seen in tendinosis tendon tissue.     

Studies on the Characterization of the Subtype(S) of Muscarinic Receptor Involved in Prostacyclin Synthesis in Rabbit Cardiomyocytes

Abstract

The present study was conducted to localize and characterize the subtype(s) of muscarinic receptor involved in prostacyclin (PGI₂) production elicited by the cholinergic transmitter acetylcholine (ACh) in various cell types in the rabbit heart. ACh increased PGI₂ synthesis measured as 6-keto-PGF1α, in cultured coronary endothelial cells and freshly dissociated ventricular myocytes in a dose dependent manner but not in cultured coronary smooth muscle cells of rabbit heart. McN-A-343, a partially selective M₁ muscarinic ACh receptor (mAChR) agonist, did not alter 6-keto-PGF1α synthesis in these cell types. ACh induced 6-keto-PGF1α synthesis in coronary endothelial cells and ventricular myocytes was not altered by a low concentration (10^?8 M) of pirenzipine, an M₁ mAChR antagonist but was reduced by a higher concentration (10^?6 M). In coronary endothelial cells ACh induced 6-keto-PGF1α production was reduced by hexahydro-sila-difendial (HHSiD), an M₃ mAChR antagonist, and in ventricular myocytes by both 11-(2-[(di-ethylamino) methyl]-1-piperidinyl]acetyl-5,11-dihydro-6-H-pyrido-[2,3-b]-benzodiazepine-6 one] (AF-DX 116), an M₂ receptor antagonist, and HHSiD. The decrease by ACh of isoporterenol stimulated cAMP accumulation was minimized by AF-DX 116 but not by HHSiD or pirenzipine. Pertussis toxin treatment minimized ACh induced decrease in isoproterenol stimulated rise in cAMP and ATP release, but not ACh induced 6-keto-PGF1α synthesis. These data suggest that ACh stimulates prostacyclin production in coronary endothelial cells via M₃ mAChR and in ventricular myocytes M₂ and M₃ mAChR. Moreover, ACh induced decrease in cAMP, but not the increase in 6-keto-PGF1α production, is mediated by pertussis toxin sensitive Gαi proteins in these cells.     

Seasonal variations in the muscarinic regulation of acetylcholine release from Torpedo electric organ nerve terminals

The cholinergic nerve endings of the electric organs of Torpedo ocellata contains presynaptic muscarinic acetylcholine receptors (mAChR) which regulate acetylcholine (ACh) release by negative feedback. The efficiency of this muscarinic regulation varies circannually: maximal inhibition is observed in the winter, much smaller effects in the fall and spring, and no effect is observed during the summer. These variations are accompanied by seasonal changes in the ability of the mAChR to trigger the synthesis of its second messenger (a prostaglandin E-like substance) and in the ability of exogenous prostaglandin E2 to inhibit ACh release. No seasonal changes were found in the number of presynaptic mAChRs. These findings suggest that the observed seasonal variations are due to changes in both the metabolism of prostaglandins in the electric organ and the sensitivity of the ACh-releasing apparatus to the muscarinic second messenger.     

The lymphocytic cholinergic system and its contribution to the regulation of immune activity

Lymphocytes express most of the cholinergic components found in the nervous system, including acetylcholine (ACh), choline acetyltransferase (ChAT), high affinity choline transporter, muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively), and acetylcholinesterase. Stimulation of T and B cells with ACh or another mAChR agonist elicits intracellular Ca2+ signaling, up-regulation of c-fos expression, increased nitric oxide synthesis and IL-2-induced signal transduction, probably via M3 and M5 mAChR-mediated pathways. Acute stimulation of nAChRs with ACh or nicotine causes rapid and transient Ca2+ signaling in T and B cells, probably via alpha7 nAChR subunit-mediated pathways. Chronic nicotine stimulation, by contrast, down-regulates nAChR expression and suppresses T cell activity. Activation of T cells with phytohemagglutinin or antibodies against cell surface molecules enhances lymphocytic cholinergic transmission by activating expression of ChAT and M5 mAChR, which is suggestive of local cholinergic regulation of immune system activity. This idea is supported by the facts that lymphocytic cholinergic activity reflects well the changes in immune system function seen in animal models of immune deficiency and immune acceleration. Collectively, these data provide a compelling picture in which lymphocytes constitute a cholinergic system that is independent of cholinergic nerves, and which is involved in the regulation of immune function.     

麻醉剂氟烷对心脏毒蕈碱型钾通道的影响

神经递质乙酰胆碱（ＡＣｈ）调节心脏功能最重要的离子通道就暗毒蕈碱型钾通道（ｉＫ,ＡＣｈ）,该通道由ＡＣｈ经鸟苷酸调节蛋白（Ｇ蛋白）的βγ亚单位而激活。本实验彩全细胞膜片箝方法,观察了麻醉药氟烷对豚鼠心房肌细胞ｉＫ,ＡＣｈ的影响。氟烷对ｉＫ,ＡＣｈ电流具抑制效应,灌注之后可使ＡＣｈ激活的ｉＫ,ＡＣｈ速率减慢,峰植下降。但其抑制ｉＫ,ＡＣｈ的程度依激活方式而异：经正常激活途径,即由ＡＣｈ激活毒蕈碱Ｍ样     

Characterization of the m4 muscarinic receptor Ca2+ response in a subclone of PC-12 cells by single cell flow cytometry. Inhibition of the response by bradykinin

We have studied Ca2+ mobilization mediated by the constitutively expressed muscarinic receptor on a subclone of PC-12 cells. The subclone, ACH2, was isolated with a flow cytometer by selection of single cells that exhibited a strong intracellular Ca2+ response to acetylcholine (ACh). Cell to cell heterogeneity of resting Ca2+ levels was markedly reduced in the subclone and homogeneity of the population response was also dramatically improved. ACH2 cells were highly sensitive to ACh and the Ca2+ response in all cells was blocked by muscarinic antagonists. Membranes from ACH2 exhibited muscarinic binding affinities which were not typical of M1, M2, or M3 receptors but were consistent with the profile of the putative m4 receptor. The same percentage of cells responded to ACh whether or not extracellular Ca2+ was reduced with EGTA, but the response was eliminated in all cells by preincubation with pertussis toxin. Thus, the constitutive m4 receptor on ACH2 cells is efficiently coupled to intracellular Ca2+ release by a pertussis toxin-sensitive mechanism. Stimulation of the ACH2 cells by bradykinin (BK) evoked a Ca2+ response in 90% of the cells. Prestimulation with BK diminished the magnitude of the muscarinic Ca2+ response but did not reduce the number of cells which responded to ACh. Inhibition was partially attributed to inhibition of a Ca2+ influx pathway in resting cells. Thus, the signaling mechanism coupled to the m4 muscarinic receptor can be inhibited by signals initiated by the BK receptor.     

N-ethylmaleimide uncouples muscarinic receptors from acetylcholine- sensitive potassium channels in bullfrog atrium

The effect of N-ethylmaleimide (NEM), a sulphydryl alkylating agent, on the acetylcholine-activated K+ current, IK(ACh), has been studied in single cells from bullfrog atrium using a tight-seal, whole-cell voltage clamp technique. Addition of NEM (5 x 10(-5) M) produced a time-dependent complete block of IK(ACh). Dialysis of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S, 5-10 x 10(-4) M), a nonhydrolyzable GTP analogue, into the myoplasm from the recording pipette gradually activated IK(ACh) even in the absence of acetylcholine. This effect is thought to be due to a GTP gamma S-induced dissociation of GTP-binding proteins (Gi and/or Go) into subunits that can directly activate these K+ channels. When NEM (5 x 10(-5) M) was applied after the GTP gamma S effect had fully developed, it failed to inhibit the GTP gamma S-induced K+ current, indicating that the NEM effect is unlikely to be on the dissociated subunits of the GTP-binding protein(s) or on the K+ channels. In contrast, pretreatment with NEM before GTP gamma S application markedly reduced the muscarinic K+ current, suggesting that NEM can block this K+ current by inhibition of the dissociation of the GTP-binding proteins into functional subunits. In NEM-treated cells the stimulatory effect of isoproterenol on ICa was present, but the inhibitory action of ACh on ICa was completely abolished. These results demonstrated that NEM can preferentially inhibit muscarinic receptor-effector interactions, probably by alkylating the GTP-binding proteins that are essential for these responses.     

Ontogenesis of physiological responsiveness and guanine nucleotide sensitivity of cardiac muscarinic receptors during chick embryonic development

Atria isolated from 4-day chick embryos were much less responsive to the negative chronotropic effect of muscarinic agonists than were atria from 5- or 8-day embryos, even though the density of muscarinic acetylcholine receptors (mAChR) was similar at all these ages. The mAChR in hearts from 4-day embryos were also significantly less susceptible to regulation of receptor number by in vivo agonist treatment and required a 2-5-fold greater dose of the muscarinic agonist carbachol to achieve a decrease in receptor number equivalent to that observed in 5- or 8-day embryonic hearts. When 4-day atrial membranes were assayed in physiological buffers, agonist binding to the mAChR was not regulated by GTP unless a sulfhydryl reducing agent was present. Receptors from 5- and 8-day embryos did not require addition of a sulfhydryl reducing agent in order to see guanine nucleotide effects on agonist binding. Even in the presence of a sulfhydryl reducing agent, carbachol binding to the mAChR in 4-day membranes was much less sensitive to guanyl-5'-yl imidodiphosphate (GppNHp) than binding to mAChR in 5- or 8-day membranes. In addition, forskolin-activated adenylate cyclase activity was much less sensitive to inhibition by GppNHp in membranes from 4-day atria than from 5- and 8-day atria. The GTP-binding component (NI) which couples the mAChR to inhibition of adenylate cyclase activity was examined by covalent modification with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Embryonic chick heart expresses multiple muscarinic acetylcholine receptor subtypes. Isolation and characterization of a gene encoding a novel m2 muscarinic acetylcholine receptor with high affinity for pirenzepine.

Muscarinic acetylcholine receptors (mAChR) are G protein-coupled receptors which are highly conserved across mammalian species. Chick cardiac mAChR, however, have been shown to be pharmacologically, immunologically, and biochemically distinct from m2 mAChR expressed in mammalian heart. We previously reported the isolation and characterization of a novel chicken mAChR, cm4, which is expressed in chick heart and brain. We report here the isolation of an additional chicken mAChR gene whose deduced amino acid sequence is most homologous to the mammalian m2 receptor. Northern blot analysis demonstrated that this chicken m2 gene is also expressed in chick heart and brain. When stably transfected into Chinese hamster ovary (CHO) cells and Y1 adrenal carcinoma cells, the chicken m2 gene expresses a receptor protein which exhibits high affinity binding for the muscarinic antagonist quinuclidinyl benzilate and atropine, as well as the M1-selective antagonist pirenzepine and the M2-selective antagonist AF-DX 116. Therefore, when expressed in two heterologous cell lines, the chick m2 receptor has pharmacological properties that are similar to the chick m4 receptor as well as those reported for endogenous mAChR in chick cardiac cells. Consistent with the properties of the chick m4, as well as mammalian m2 and m4 receptors, the chick m2 receptor was able to functionally couple to both the inhibition of adenylate cyclase and the stimulation of phosphoinositide metabolism when expressed in CHO cells, but only the inhibition of adenylate cyclase when expressed in Y1 cells. We conclude from this study that the embryonic chick heart expresses multiple subtypes of mAChR which are highly conserved with their mammalian counterparts. Furthermore, the high degree of conservation between the mammalian m2 and the chick m2 muscarinic receptors suggests that the pharmacological differences that exist between these receptors are due to a relatively small number of specific amino acid changes rather than larger changes in receptor sequence or structure.     

Acetylcholine induces mesenchymal stem cell migration via Ca2+ /PKC/ERK1/2 signal pathway

Acetylcholine (ACh) plays an important role in neural and non-neural function, but its role in mesenchymal stem cell (MSC) migration remains to be determined. In the present study, we have found that ACh induces MSC migration via muscarinic acetylcholine receptors (mAChRs). Among several mAChRs, MSCs express mAChR subtype 1 (m1AChR). ACh induces MSC migration via interaction with mAChR1. MEK1/2 inhibitor PD98059 blocks ERK1/2 phosphorylation while partially inhibiting the ACh-induced MSC migration. InsP3Rs inhibitor 2-APB that inhibits MAPK/ERK phosphorylation completely blocks ACh-mediated MSC migration. Interestingly, intracellular Ca(2+) ATPase-specific inhibitor thapsigargin also completely blocks ACh-induced MSC migration through the depletion of intracellular Ca(2+) storage. PKCα or PKCβ inhibitor or their siRNAs only partially inhibit ACh-induced MSC migration, but PKC-ζ siRNA completely inhibits ACh-induced MSC migration via blocking ERK1/2 phosphorylation. These results indicate that ACh induces MSC migration via Ca(2+), PKC, and ERK1/2 signal pathways.     

Control of the cardiac muscarinic K+ channel by beta-arrestin 2

Control of the cardiac muscarinic K(+) current (i(K,ACh)) by beta-arrestin 2 has been studied. In Chinese hamster ovary cells transfected with m2 muscarinic receptor, muscarinic K(+) channel, receptor kinase (GRK2), and beta-arrestin 2, desensitization of i(K,ACh) during a 3-min application of 10 micrometer ACh was significantly increased as compared with that in cells transfected with receptor, channel, and GRK2 only (fade in current increased from 45 to 78%). The effect of beta-arrestin 2 was lost if cells were not co-transfected with GRK2. Resensitization (recovery from desensitization) of i(K,ACh) in cells transfected with beta-arrestin 2 was significantly slowed (time constant increased from 34 to 232 s). Activation and deactivation of i(K,ACh) on application and wash-off of ACh in cells transfected with beta-arrestin 2 were significantly slowed from 0.9 to 3.1 s (time to half peak i(K,ACh)) and from 6.2 to 13.8 s (time to half-deactivation), respectively. In cells transfected with a constitutively active beta-arrestin 2 mutant, desensitization occurred in the absence of agonist (peak current significantly decreased from 0.4 +/- 0.05 to 0.1 +/- 0.01 nA). We conclude that beta-arrestin 2 has the potential to play a major role in desensitization and other aspects of the functioning of the muscarinic K(+) channel.     

Islet activating protein inhibits physiological responses evoked by cardiac muscarinic acetylcholine receptors. Role of guanosine triphosphate binding proteins in regulation of potassium permeability

The involvement of GTP binding proteins in muscarinic acetylcholine receptor (mAChR) mediated responses of cultured chick embryonic cardiac muscle cells was studied by using islet activating protein (IAP) from Bordetella pertussis. Incubation of cells for 24 h with IAP resulted in inhibition of subsequent IAP-catalyzed incorporation of [alpha-32P]ADP-ribose into membrane proteins of Mr 39 000 (No alpha) and 41 000 (Ni alpha); treatment of cultures with 5 ng/mL IAP was sufficient to ADP-ribosylate all available No alpha and Ni alpha. Inhibition of forskolin-stimulated cAMP accumulation by the muscarinic agonist carbachol was abolished in cultures pretreated with IAP. The affinity of carbachol for the mAChR in membranes from IAP-treated cells was considerably decreased compared to control membranes and was not further decreased by addition of guanyl-5'-yl imidodiphosphate. In contrast, the affinity of carbachol for the mAChR on intact cells was not affected by pretreatment with IAP. To investigate the involvement of No and/or Ni in mAChR-mediated increases in K+ permeability, the effect of IAP treatment on mAChR stimulation of 86Rb+ efflux was determined. Treatment of cultures with 5 ng/mL IAP for 24 h completely blocked the stimulation of 86Rb+ efflux evoked by carbachol. Because previous work has shown that mAChR regulation of K+ permeability is independent of changes in cAMP levels, these results suggest a role for No and/or Ni in coupling the mAChR directly to K+ channels in the heart.     

Disruption of cytoskeletal integrity impairs Gi-mediated signaling due to displacement of Gi proteins

beta1 integrins play a crucial role as cytoskeletal anchorage proteins. In this study, the coupling of the cytoskeleton and intracellular signaling pathways was investigated in beta1 integrin deficient (-/-) embryonic stem cells. Muscarinic inhibition of the L-type Ca2+ current (ICa) and activation of the acetylcholine-activated K+ current (IK,ACh) was found to be absent in beta1 integrin-/- cardiomyocytes. Conversely, beta adrenoceptor-mediated modulation of ICa was unaffected by the absence of beta1 integrins. This defect in muscarinic signaling was due to defective G protein coupling. This was supported by deconvolution microscopy, which demonstrated that Gi exhibited an atypical subcellular distribution in the beta1 integrin-/- cardiomyocytes. A critical role of the cytoskeleton was further demonstrated using cytochalasin D, which displaced Gi and impaired muscarinic signaling. We conclude that cytoskeletal integrity is required for correct localization and function of Gi-associated signaling microdomains.     

Phosphorylation of chick heart muscarinic cholinergic receptors by the beta-adrenergic receptor kinase

Previous studies have demonstrated that muscarinic cholinergic receptors (mAChR) become markedly phosphorylated when intact cardiac cells are stimulated with a muscarinic agonist. This process appears to be related to the process of receptor desensitization. However, the mechanism of agonist-induced phosphorylation of mAChR is not known. In situ phosphorylation studies suggested that agonist-induced phosphorylation of mAChR may involve the participation of a receptor-specific kinase and/or require agonist occupancy. These observations regarding phosphorylation and desensitization of mAChR are similar to observations made for beta-adrenergic receptors. Recent studies have indicated that homologous desensitization of beta-adrenergic receptors may be due to the phosphorylation of these receptors by a novel protein kinase that only recognizes the agonist-occupied form of the receptors. As muscarinic receptors are structurally homologous to beta-adrenergic receptors, we have initiated studies to identify the protein kinase responsible for the phosphorylation of muscarinic receptors by determining whether the chick heart muscarinic receptor would serve as a substrate for the beta-adrenergic receptor kinase (beta-AR kinase). We report that the purified and reconstituted chick heart muscarinic receptor serves as an excellent substrate in vitro for the beta-AR kinase. Phosphorylation of mAChR receptors by the beta-AR kinase was only observed in the presence of a muscarinic receptor agonist and was prevented in the presence of antagonist. Both the extent of phosphorylation (3-4 mol of P/mol of receptor) and the phosphoamino acid composition of the mAChR after incubation in vitro with beta-AR kinase were similar to the characteristics of agonist-induced phosphorylation of mAChR in situ.(ABSTRACT TRUNCATED AT 250 WORDS)