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A ganglionic stimulant, 1,1-dimethyl-4-phenylpiperazinium, caused both cholinergic and adrenergic responses in the isolated mouse atrium
European Journal of Pharmacology 704 (2013) 7–14
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
A ganglionic stimulant, 1,1-dimethyl-4-phenylpiperazinium, caused both cholinergic and adrenergic responses in the isolated mouse atrium Kenta Ochi a, Hiroki Teraoka a, Toshihiro Unno b, Sei-ichi Komori b, Masahisa Yamada c, Takio Kitazawa a,d,n a
Department of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan Laboratory of Pharmacology, Faculty of Applied Biological Science, Gifu University, Gifu 501-1193, Japan c Common Resources Group, Okinawa Institute of Science and Technology, Okinawa 904-0411, Japan d Department of Veterinary Science, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan b
a r t i c l e i n f o
abstract
Article history: Received 26 September 2012 Received in revised form 1 February 2013 Accepted 7 February 2013 Available online 24 February 2013
An isolated atrial preparation of the mouse is useful for analyzing the actions of drugs on the myocardium, autonomic neurons and endocardial endothelium. The aim of the present study was to examine the functions of intrinsic neurons of the atrium using a ganglionic stimulant, 1,1-dimethyl-4phenylpiperazinium (DMPP). DMPP (1–100 mM) caused a negative chronotropic action followed by a positive chronotropic action in spontaneously beating right atria and also caused biphasic inotropic actions consisting of initial inhibition followed by potentiation of electrical field stimulation (EFS)induced contraction in the left atria. Inotropic actions in the left atria induced by DMPP were characterized using some autonomic drugs and M2 and/or M3 muscarinic receptor knockout (M2RKO, M3R-KO and M2M3R-KO) mice. Atropine and hexamethonium decreased the initial negative inotropic actions of DMPP. In the atria from pertussis toxin-treated, M2R-KO and M2/M3R-KO mice, the negative inotropic actions were abolished. On the other hand, the following positive inotropic actions were decreased by hexamethonium, atropine and atenolol. In the atria from reserpine-treated mice, positive inotropic actions were also decreased. The positive inotropic action induced by DMPP was almost the same in M2R-KO mice but was reduced in both M3R-KO mice and M2/M3R-KO mice. In conclusion, DMPP caused biphasic inotropic/chronotropic actions in the mouse atrium through activation of intrinsic cholinergic and adrenergic neurons. M2 and M3 muscarinic receptors and b1-adrenoceptor are thought to be involved in these actions. & 2013 Elsevier B.V. All rights reserved.
Keywords: Mouse atrium Nicotinic receptor Intrinsic neurons Cholinergic action Adrenergic action
1. Introduction Cardiac functions (contraction force, heart rate and conduction velocity) are regulated by sympathetic and parasympathetic neurons. Activation of sympathetic neurons stimulates cardiac function through release of noradrenaline, while activation of parasympathetic neurons decreases cardiac contraction and heart rate through release of acetylcholine. In general, sympathetic preganglionic fibers make their synapses near the spinal cord and postganglionic fibers arising from the ganglia innervate cardiac tissue, whereas the parasympathetic nervous system has its ganglia within organs (intrinsic cardiac ganglia, Levy and Martin, 1981; Loffelholz and Pappano, 1985). Intrinsic cardiac ganglia were initially regarded as simple relay stations in parasympathetic n Corresponding author at: Department of Veterinary Science, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan. Tel.: þ81 11 388 4795; fax: þ 81 11 387 5890. E-mail address: [email protected] (T. Kitazawa).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.02.019
preganglionic cholinergic neurons to postganglionic cholinergic neurons innervating the myocardium. However, cardiac ganglia have been demonstrated to have a complex neurochemical phenotype different from the classical view of postganglionic cholinergic neurons. Morphological study indicated that some cholinergic nerve cell bodies contained noradrenergic neurons markers, such as tyrosine hydroxylase, dopamine-b-hydroxylase and noradrenaline transporters. The presence of such hybrid intrinsic cardiac neurons has been reported in several animal species (Baluk and Gabella, 1990; Hoard et al., 2007, 2008; Hoover et al., 2009; Rysevaite et al., 2011; Weihe et al., 2005). Therefore, regulation of cardiac contractility through these kinds of intrinsic neurons might be interesting, but the functional role of these neurons has not been clarified in detail. The isolated atrium is widely used to examine inotropic and chronotropic actions of bioactive substances. Using mice atria, we demonstrated that carbachol caused biphasic inotropic actions consisting of initial negative actions (M2 muscarinic receptor) followed by slowly developed positive actions (M3 muscarinic
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receptor, Kitazawa et al., 2009). M3 receptors were demonstrated to be expressed on the endocardial endothelium, and cyclooxygenase-2/prostaglandins pathways were shown to be involved in downstream of M3 receptor activation (Harada et al., 2012). Analysis of electrical field stimulation (EFS)-induced responses of isolated visceral organs has been used to identify functional innervation. For example, analysis of EFS-induced responses indicated functional innervation of the gastrointestinal tract (Burnstock, 1972). Similarly, nitrergic nerves were demonstrated in blood vessels using EFS (Toda and Okamura, 2003). EFS has also been applied to isolated atria of rats and guinea-pigs, and characterization of evoked responses suggested inhibitory cholinergic and excitatory adrenergic innervation of cardiac tissues (Goto et al., 1987; Saito et al., 1986). However, EFS can excite all neural components in cardiac tissues (intrinsic neurons, efferent and sensory neurons) comprehensively and, therefore, EFS might not be a suitable stimulation for analyzing the function of intrinsic neurons in the mouse heart. Effects of ganglionic stimulants on the heart rate of rodents in vitro have been reported. However, the responses have not been characterized in detail (Nakatani et al., 1994; Wong, 1994). The present study was designed to elucidate the function of cardiac intrinsic neurons in the mouse atrium. To accomplish the objectives, intrinsic neurons expressing the nicotinic receptor were excited by a ganglionic stimulant, 1,1-dimethyl-4-phenylpiperazinium (DMPP), and the evoked mechanical actions were characterized. Atria from reserpine- or pertussis toxin-treated mice and muscarinic receptor knockout mice were also used for further characterization of the DMPP-induced responses.
2. Materials and methods 2.1. Animals and tissue preparations All experiments were performed in accordance with the institutional guidelines approved by the Animal Ethics Committee of Rakuno Gakuen University, Ebetsu, Hokkaido, Japan. Wild-type mice (DDY strain; Sankyo Lab Service, Sapporo, Japan) and mice lacking either the M2 muscarinic receptor (M2R-KO) or the M3 muscarinic receptor (M3R-KO) or both M2 and M3 muscarinic receptors (M2/M3R-KO) were used in the present experiments (either male or female). The generation of M2R-KO, M3R-KO and M2/M3R-KO mice has been described previously (Gomeza et al., 1999; Struckmann et al., 2003; Yamada et al., 2001). The genetic backgrounds of the mice used in the present study were 129J1 (50%) CF1 (50%) for M2R-KO mice, 129vEv (50%) CF1 (50%) for M3R-KO mice, and 129J1 (25%) 129SvEv (25%) CF1 (50%) for M2/M3R-KO mice. The animals were housed in ventilated-polycarbonate cages. The temperature of the animal room was maintained at 2371 1C with relative humidity of 40%–60% and a daily light/dark cycle (7:00 am–7:00 pm). Food (CRF-1, Oriental Yeast Co Ltd, Japan) and water were given ad libitum. Adult mice that were more than 3 months old (weight: 23–30 g) were killed by cervical dislocation. The beating heart was isolated from each animal and immersed in warmed and bubbling Krebs solution (NaCl, 118 mM; KCl, 4.75 mM; MgSO4, 1.2 mM; KH2PO4, 1.2 mM; CaCl2, 2.5 mM; NaHCO2, 25 mM; and glucose, 11.5 mM, 37 1C, gassed with 95% O2 þ5% CO2). To induce myocardial contraction, the left atrium was placed between a pair of platinum rod electrodes and suspended vertically in an organ bath. The end of the preparation was tied and connected to a force–displacement transducer. EFS (1 Hz, 2 ms in duration, 1.5 threshold voltage; Kitazawa et al., 2009; Harada et al., 2012) was applied by an electrical stimulator. After steady EFS-induced
contraction had been established, several drugs including DMPP were applied to the organ bath and the evoked responses were observed. In experiments for observation of chronotropic action, one end of the beating right atrium was attached with thread to a stationary glass rod, and the opposite end was tied with thread to a force–displacement transducer to record spontaneous contraction. 2.2. Experimental protocols Inotropic actions: After establishment of steady EFS-induced contraction (generally 60–70 min of equilibration time) of the left atrium, DMPP (1–100 mM) was added to the organ bath and its effects on EFS-induced contraction was observed for 15 min. After determining the concentration–response relationships of DMPP, pharmacological properties of the DMPP-induced inotropic actions were characterized using several autonomic drugs. Since the inotropic actions induced by DMPP (100 mM) were not reproducible even at 1-h-interval application in this experimental condition, DMPP was applied only one time in one preparation. In brief, atrial preparations were treated with respective autonomic drugs for 15 min and then DMPP was applied and its inotropic action was compared with that in non-treated atria (control). To examine the involvement of the M2 muscarinic receptor in DMPPinduced actions, DDY mice were treated with pertussis toxin (300 mg/kg, i.p.) 96 h prior to experiments. Pertussis toxin has been shown to abolish M2 receptor-mediated negative chronotropic and inotropic actions in the mouse atrium though inhibition of Gi protein function (Kitazawa et al., 2009). Treatment with pertussis toxin did not cause any obvious behavioral changes or toxic effects. After 96 h, atria were isolated and the inotropic actions induced by DMPP were compared with those in control atria from non-treated mice. Involvement of adrenergic neurons in DMPP-induced actions was examined using atria from reserpine-treated mice. Reserpine inhibits adrenergic neural function through depletion of noradrenaline contents (Martı´nezOlivares et al., 2006). Reserpine was injected (10 mg/kg, i.p.) and atria were isolated 18 h later, and the effects of DMPP were compared with the control. Catalepsy was observed in all mice treated with reserpine. DMPP-induced inotropic actions were also examined in atria from muscarinic receptor knockout mice (M2RKO, M3R-KO and M2/M3R-KO mice), and muscarinic receptor subtypes involved in the actions were characterized. Chronotropic actions: Spontaneously beating right atria from control (non-treated) mice, pertussis toxin-treated mice, reserpinetreated mice and muscarinic receptor knockout mice were suspended vertically in an organ bath and equilibrated for 1 h. DMPP (1–100 mM) was applied, and its effects on both heart rate and contraction amplitude were analyzed as chronotropic and inotropic actions. Similar to the case of the left atrium, the DMPP-induced chronotropic action at 100 mM was not reproducible at 1-h-interval application. Therefore, 100 mM DMPP was applied one time in each atrium for characterization of pharmacological properties. In brief, atrial preparations were treated with respective autonomic drugs for 15 min and then DMPP (100 mM) was applied to compare the induced actions in the presence and absence of drugs. 2.3. Chemicals The following chemicals were used: Atenolol (Sigma), atropine sulfate (Wako Pure Chemicals, Osaka, Japan), 1,1-dimethyl-4phenylpiperazinium iodide (DMPP, Sigma), hexamethonium chloride dehydrate (Wako), physostigmine sulfate (Wako), reserpine (Apoplon, Daiichi Sankyo, Tokyo, Japan) and pertussis toxin (Wako). Pertussis toxin was dissolved in sterilized saline and was
K. Ochi et al. / European Journal of Pharmacology 704 (2013) 7–14
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Fig. 1. Effects of various concentrations of DMPP on heart rate and amplitude of contraction in spontaneously beating right atria of mice. DMPP (1, 10, 30, 100 mM) was applied to an organ bath in which a spontaneously beating right atrium was suspended, and changes in heart rate (A, B) and contraction amplitude (C, D) were examined. Initial negative chronotropic (B) and negative inotropic actions (D) observed within 60 s are highlighted. Ordinate: relative heart rate and amplitude of contraction (absence of DMPP¼ 100%). Values are mean 7 S.E.M. of more than four experiments.
given to mice (300 mg/kg, i.p.) in a volume of 0.1 ml/10 g body weight. Reserpine was also given to mice (10 mg/kg, i.p.).
3.2. Pharmacological properties of DMPP-induced actions in the right atria
2.4. Statistics
Since positive and negative inotropic/chronotropic actions in the right atria were marked at 100 mM DMPP, this concentration was used to determine the pharmacological properties. First, the effects of some autonomic drugs on the transient negative chronotropic actions were examined (Fig. 2A). Atropine (1 mM) or hexamethonium (100 mM) significantly decreased the transient negative chronotropic actions. On the other hand, atenolol (1 mM) did not change the actions. DMPP-induced actions were compared in the right atria from pertussis toxin- and reserpinetreated mice. These treatments did not affect beating rate of isolated atria (pertussis toxin: 335 712 beats/min, n ¼4; reserpine: 367735 beats/min, n ¼4). DMPP-induced negative chronotroic actions were abolished in the atria from pertussis toxintreated mice but did not change in the atria of reserpine-treated mice (Fig. 2A). In the atria of reserpine-treated mice, negative chronotropic actions lasted a long time compared with that in control mice (relative heart rates at 20 s, 30 s and 40 s after application, control: 10275.5%, 13077.6% and 13477%, n¼6; reserpine: 7378%, 8476% and 9974.5%, n¼4). In the atria from muscarinic receptor knockout mice, heart rate was not significantly different from that of control mice (M2R-KO: 335734 beats/min, n¼ 5; M3R-KO: 356733 beats/min, n¼5; M2/M3R-KO: 314737 beats/ min, n¼5). Negative chronotropic action of DMPP was abolished and, reversibly, positive chronotropic action was observed at 10 s in M2R-KO mice (189710%, n¼5) and M2/M3R-KO mice (17172.5%, n¼5). In the atria of M3R-KO mice, the DMPP-induced action (10974.3%, n¼5) was not different from that in control (wild-type) mice (Fig. 3A). Pharmacological properties of the DMPP (100 mM)-induced negative inotropic actions were examined (Fig. 2C). The transient negative inotropic actions were inhibited significantly by atropine (1 mM) and hexamethonium (100 mM). However atenolol (1 mM)
Data are expressed as means7 S.E.M of at least four independent experiments using left and right atria isolated from different mice. Students’ t-test or one-way analysis of variance (ANOVA) followed by Dunnett’s test was used for analysis of the effects of drugs on actions of DMPP. One-way ANOVA followed by Bonferroni’s multiple comparison test was used to compare the actions of DMPP among muscarinic receptor knockout mice. Po0.05 was considered to be statistically significant.
3. Results 3.1. Effects of DMPP in spontaneously beating right atria Spontaneous contraction of the right atria was 351720 beatsmin (n¼ 8) in the present experiments. The effects of DMPP on heart rate and amplitude of spontaneous contraction are shown in Fig. 1. One mM DMPP did not cause any change in heart rate or amplitude of contraction. However, higher concentrations of DMPP (10–100 mM) increased heart rate in a concentrationdependent manner, and the positive chronotropic actions lasted for up to 5 min. However, when 100 mM DMPP was applied, a transient decrease in heart rate (relative heart rate¼9171.8%, n ¼6) was observed from 5 to 15 s after application (Fig. 1B). DMPP (10–100 mM) also affected the amplitude of spontaneous contraction. Ten mM DMPP caused only small positive inotropic actions, but 30 or 100 mM DMPP caused transient negative inotropic actions (at 10 s, relative amplitude, 30 mM¼80 79.3%, n ¼5; 100 mM¼7476.6%, n¼5) followed by positive inotropic actions (Fig. 1C and D).
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Fig. 2. Pharmacological properties of DMPP (100 mM)-induced actions in spontaneously beating right atria. DMPP-induced chronotropic actions were divided into initial negative actions (A) and following positive actions (B). DMPP-induced inotropic actions were also divided into initial negative actions (C) and following positive actions (D). Effects of hexamethonium (C6, 100 mM), atropine (1 mM) and atenolol (1 mM) on DMPP-induced actions were investigated. DMPP-induced actions were also observed in the right atria from pertussis toxin (PTX)- and reserpine-treated mice. Each column indicates relative values of heart rate (chronotropic action, A, B) and contraction amplitude (inotropic action, C, D). Values are means7 S.E.M. of more than four experiments. *Po 0.05, **Po 0.01. Significantly different from the control response of DMPP (black column).
did not change the actions. The negative inotropic actions were not decreased but tended to be potentiated in the atria of reserpine-treated mice. In the atria of pertussis toxin-treated mice, the inhibition of amplitude by DMPP was abolished (Fig. 2C). Comparison of the inotropic actions induced by DMPP in the muscarinic receptor knockout mice indicated that the negative inotropic action was unchanged in the atria from M3R-KO mice (80716%, n ¼5) but significantly decreased and changed to positive inotropic responses in the atria from M2R-KO mice (129711%, n ¼5) and M2/M3R-KO mice (12075.7%, n ¼5) (Fig. 3B). The DMPP-induced positive chronotropic and positive inotropic actions were also characterized. On the basis of the time course of DMPP-induced responses, chronotropic action was determined at 60 s and inotropic action was determined at 45 s after application (Fig. 1A and C). Positive chronotropic actions were not changed by atropine but were significantly decreased by hexamethonium (100 mM) and atenolol (1 mM). In the atria from reserpine-treated mice, positive chronotropic actions of DMPP were abolished, but pertussis toxin treatment was ineffective in decreasing the positive chronotropic actions (Fig. 2B). Among muscarinic receptor knockout mice, positive chronotropic action was significantly enhanced in the atria from M2R-KO mice (215716%, n¼5) and M2/M3R-KO mice (226 730.6%, n¼5) but was almost the same in atria from M3R-KO mice (13875.4%, n ¼5) (Fig. 3A). Next, the DMPP-induced positive inotropic actions were characterized. Relative amplitude of spontaneous contraction at 45 s was 154 77.0% (n¼5), and this action was significantly decreased by hexamethonium (100 mM) and atenolol (1 mM). However, atropine (1 mM) was ineffective in decreasing
the inotropic action. The positive inotropic action was abolished in the atria from reserpine-treated mice but was almost the same in the atria from pertussis toxin-treated mice (Fig. 2D). The DMPP-induced inotropic actions were compared among muscarinic receptor knockout mice. The relative amplitudes of spontaneous contraction at 45 s were almost the same in the atria of M2R-KO, M3R-KO and M2/M3R-KO mice: 153 719% (n ¼5), 14675.6% (n¼ 5) and 139 717.5% (n ¼5), respectively (Fig. 3B).
3.3. Effect of DMPP on EFS-induced contraction of left atria After observing the reproducible EFS-induced contraction, DMPP (1–100 mM) was applied in an organ bath and change in the amplitude of contraction was investigated. DMPP caused a transient small negative inotropic action followed by long-lasting positive inotropic action. The negative inotropic action was shortlived and was not observed after 30 s of application. The relative amplitudes of EFS-induced contraction at 15 and 30 s were 10171.3% and 10273% (1 mM, n ¼4), 100 71% and 10574.8% (10 mM, n ¼7), 96.4 71.9% and 10772.8% (30 mM, n¼9), and 9272.2% and 11777.4% (100 mM, n ¼7), respectively, indicating concentration-dependency of negative inotropic actions (Figs. 4 and 5). The time course of DMPP-induced inotropic actions indicated that the maximum responses occurred at about 3–7 min after application and that the maximum inotropic actions were 10271.4% (1 mM, n¼4), 11873.6% (10 mM, n¼7), 13076.1% (30 mM, n¼9), and 18677.4% (100 mM, n¼7). These positive inotropic responses were observed even at 15 min after application (Fig. 5).
K. Ochi et al. / European Journal of Pharmacology 704 (2013) 7–14
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Fig. 4. Effects of DMPP on EFS-induced contraction of isolated left atria of mice. Each trace indicates a typical inotropic action of DMPP (100 mM) in the absence (control) and presence of hexamethonium (C6, 100 mM), atropine (1 mM) and atenolol (1 mM). Left atria were isolated from pertussis toxin (PTX)- and reserpinetreated mice, and DMPP-induced actions were also examined. DMPP was applied in an organ bath at the time point indicated by an arrow (.).
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Time (sec) Fig. 3. Comparison of DMPP (100 mM)-induced actions on heart rate and amplitude of spontaneous contraction in right atria isolated from muscarinic receptor knockout mice. Each graph shows the time courses of DMPP (100 mM)-induced chronotropic action (A) and inotropic action (B) observed in wild-type (control), M2R-KO, M3R-KO and M2/M3R-KO mice. Ordinate: relative heart rate and amplitude of spontaneous contraction (absence of DMPP ¼100%). Statistical comparison was performed as described in the text (see results). Values are means7 S.E.M. of more than four experiments.
3.4. Pharmacological properties of DMPP-induced actions in the left atria Negative inotropic actions of DMPP (100 mM) were decreased by treatment with atropine (1 mM) or hexamethonium (100 mM) but tended to be enhanced by atenolol (1 mM). In the atria from pertussis toxin-treated mice, DMPP-induced negative inotropic action was decreased significantly (Figs. 4 and 6A). Next, the maximum positive inotropic responses were compared in the absence and presence of autonomic drugs. The positive inotropic action of DMPP was reduced by atropine (1 mM) or hexamethonium (100 mM) through decreasing onset of the responses. Atenolol (1 mM) and reserpine-treatment were also effective in decreasing the positive inotropic actions. Inhibition of the positive inotropic actions by reserpine was partial and different from the case in the right atria (Figs. 4 and 6B). The inotropic actions induced by DMPP were compared in the atria from muscarinic receptor knockout mice (Figs. 7 and 8). The initial inhibition of EFS-induced contraction was abolished in the atria from M2R-KO and M2/M3R-KO mice. The relative
amplitudes of EFS-induced contraction at 15 s were 10773% (n¼7) in M2R-KO mice, 9471.8% (n¼4) in M3R-KO mice and 10071.1% (n¼6) in M2/M3R-KO mice. On the other hand, positive inotropic actions were significantly decreased in the atria from M3RKO and M2/M3R-KO mice but not in the atria from M2R-KO mice. A significant difference was also found between the values of M2R-KO mice and M2/M3R-KO mice (Figs. 7 and 8).
3.5. Inotropic actions of atropine, physostigmine and atenolol During the present experiments, it was observed that atropine itself caused slight changes in amplitude of the EFS-induced contraction. It was speculated that intrinsic nerves were also stimulated by EFS. Therefore, we examined the effects of cholinergic and adrenergic drugs on the EFS-induced contraction. Atropine (1 mM) slowly potentiated EFS-induced contraction, and the response reached a peak after 5 min of application (12174.6%, n ¼6). On the other hand, physostigmine (300 nM) decreased the EFS-induced contraction. Relative amplitude of EFS-induced contraction was 60.874.1% (n ¼5) at 10 min after application, but physostigimine-induced inhibition was not observed in the atria from M2R-KO mice (9872.0%, n¼5). Atenolol (1 mM) was effective in decreasing the EFS-induced contraction. Inhibition developed slowly and reached a peak after 12 min of application (relative contraction ¼8672.4%, n ¼4), but in the atria from reserpine-treated mice, atenolol-induced inhibition disappeared (relative contraction¼ 9671.5%, n ¼4, p¼0.006 vs. control).
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Fig. 5. Concentration-dependent inotropic actions by DMPP in the left atria of mice. DMPP (1, 10, 30, 100 mM) was applied to an organ bath, and time courses of inotropic actions are indicated on a long time scale (A, 0–15 min) and short time scale (B, 0–1 min, for clarification of negative inotropic action). Ordinate: relative amplitude of contraction (absence of DMPP¼100%). Values are means7 S.E.M. of more than four experiments.
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Fig. 6. Pharmacological properties of DMPP (100 mM)-induced negative (A) and positive inotropic actions (B) in the left atria of mice. Effects of hexamethonium (C6, 100 mM), atropine (1 mM) and atenolol (1 mM) on DMPP-induced actions were investigated. DMPP-induced inotropic actions were also examined in the left atria from pertussis toxin (PTX)- and reserpine-treated mice. Each column indicates relative amplitude of EFS-induced atrial contraction (absence of DMPP ¼ 100%). Values are means7 S.E.M. of more than 4 experiments. *P o0.05, **P o0.01. Significantly different from the control response of DMPP (black column).
4. Discussion Immunohistochemical studies have revealed the neurochemical characteristics of cardiac intrinsic neurons of the mouse heart, and cholinergic neurons and cholinergic neurons containing adrenergic neural markers (tyrosine hydroxylase, dopamine-b-hydroxylase and noradrenaline transporters) have also been demonstrated (Hoard et al., 2007, 2008; Rysevaite et al., 2011). However, there have been few functional studies on these intrinsic cardiac neurons, although mice have been widely used in physiological and pharmacological studies on cardiac function, and genetically manipulated mice are available for use. This functional study showed the characteristics of cardiac intrinsic neurons in the mouse atrium. DMPP, a ganglionic stimulant, caused biphasic inotropic/chronotropic actions consisting of both cholinergic and adrenergic properties, and M2 muscarinic receptor, M3 muscarinic receptor and b1-adrenoceptor are thought to be involved in the actions of DMPP. Therefore, it is thought that intrinsic neurons with cholinergic and/or adrenergic neural properties have physiological roles in the regulation of cardiac contraction in mice. Studies on the effects of DMPP and nicotine on contractility of isolated atrial preparations have been carried out using rats, mice, guinea-pigs and rabbits (Cheav et al., 1992; Nakatani et al., 1994; Westfall and Brasted, 1972; Wong, 1994). Negative inotropic/chronotropic actions (Nakatani et al., 1994) and positive inotropic actions (Cheav et al., 1992; Westfall and Brasted, 1972; Wong, 1994) have been reported as responses to ganglionic stimulants. Adrenergic neurons were shown to be involved in the positive inotropic actions
(Cheav et al., 1992; Westfall and Brasted, 1972), but negative inotropic actions were not modified by atropine or hexamethonium and were thought to be the result of direct action on cardiac muscle cells (Nakatani et al., 1994). In an isolated blood perfused canine right atrium, nicotine caused negative chronotropic actions followed by positive chronotropic actions (Ren et al., 1991). In the isolated atria of mice, DMPP caused biphasic actions consisting of initial negative chronotropic/inotropic actions followed by long-lasting positive chronotropic/inotropic actions similar to previous results obtained in the canine right atrium (Ren et al., 1991). Negative chronotropic and inotropic actions were inhibited by hexamethonium and atropine, and these actions were not observed in the atria from pertussis toxin-treated or M2R-KO mice. Since negative inotropic/chronotropic actions of carbachol were abolished in the atria of pertussis toxintreated and M2R-KO mice (Kitazawa et al., 2009; Harada et al., 2012), the results of the present study suggested that DMPP excited nicotinic receptors of intrinsic cholinergic neurons and that released acetylcholine reduced atrial contractility through activation of M2 muscarinic receptors located on the myocardium (Harada et al., 2012). Mouse cardiac ganglia containing intrinsic cholinergic neurons have already been demonstrated morphologically, and acetylcholine elicited a hexamethonium-sensitive inward current in cholinergic neurons (Hoard et al., 2007, 2008; Rysevaite et al., 2011). Taken together, these results suggest that the physiological function of these cholinergic intrinsic neurons is to decrease heart rate and contraction. In a previous study on the mouse atrium, negative chronotropic/inotropic actions were not found (Wong, 1994). Since negative chronotropic/ inotropic actions of DMPP are weak and short-lived and are soon
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overcome by following positive chronotropic/inotropic actions, it is possible that transient negative responses were overlooked in the previous study. DMPP caused positive chronotropic actions in the right atria and positive inotropic actions in both atria, and these actions were decreased by hexamethonium, atenolol and reserpine-treatment. Since atenolol is a blocker of the cardiac b1-adrenoceptor and reserpine is an adrenergic neuron blocker, the present results indicated the involvement of adrenergic neurons in positive chronotropic/inotropic actions, consistent with the results of previous studies (Cheav et al., 1992; Westfall and Brasted, 1972). However, involvement of adrenergic neurons in the DMPP-induced actions was thought to be different between the right and left atria, because reserpine abolished the positive chronotropic/inotropic actions of DMPP in the right atria but only partially inhibited the actions in the left atria. Non-adrenergic mechanisms are suggested to be involved in the positive inotropic action of DMPP in the left atria. Immunohistochemical studies have demonstrated the presence of cholinergic, adrenergic and biphenotypic intrinsic neurons in mouse cardiac ganglia (Hoard et al., 2007, 2008; Rysevaite et al., 2011). Generally, in sympathetic neurons, the nicotinic receptor is thought to be located on the cell body and it causes a depolarization-eliciting action potential, which then triggers Ca2 þ -dependent exocytosis at the axon terminal (Boehm and Huck, 1997). Therefore, the results suggested that intrinsic adrenergic or biphenotypic neurons in mouse cardiac ganglia have a regulatory function and that released noradrenaline stimulates b1-adrenoceptors for positive inotropic and chronotropic actions. Parasympathetic vagus nerves are thought to have synapses in cardiac ganglia (Levy and Martin, 1981; Loffelholz and Pappano, 1985). The results of the present in vitro study can explain the vagal nerve-mediated tachycardia (adrenergic neurons being partially involved) observed in an in vivo study (Levy, 1994).
160
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Fig. 7. Typical inotropic actions of DMPP (100 mM) in left atria isolated from wild-type (control), M2R-KO, M3R-KO and M2M3R-KO mice. Initial negative inotropic action was abolished in the atria from M2R-KO mice and M2M3R-KO mice. Amplitude of positive inotropic action was attenuated in the atria from M3R-KO and M2M3R-KO mice.
Relative amplitude (%)
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Fig. 8. Involvement of M2 and M3 muscarinic receptors in negative and positive inotropic actions of the mouse left atrium induced by DMPP. DMPP (100 mM) was applied to an organ bath, and the evoked negative (A) and positive (B) inotropic actions (see Fig. 7) were compared. Each column indicates relative amplitude of EFS-induced atrial contraction. Values are means7 S.E.M. of more than four experiments. *P o0.05, **Po 0.01. Significantly different from the value for wildtype mice (black column). #P o0.05. Significantly different from the value for M2R-KO mice (open column).
We previously reported that M3 muscarinic receptors were involved in the carbachol-induced positive inotropic actions of the mouse left atrium through activation of the cyclooxygenase-2prostaglandin pathway (Kitazawa et al., 2009; Harada et al., 2012). In the present study, DMPP-induced inotropic action was significantly decreased in the left atria of M3R-KO and M2/M3R-KO mice but not in those of M2R-KO mice. In the atria of wild-type mice, atropine slowed down the onset of the response and inhibited the positive inotropic action of DMPP. These results indicated the involvement of M3 muscarinic receptors in the DMPP-induced positive inotropic action of the left atrium. Since a non-adrenergic positive inotropic component in the left atria was suggested to be present by the functional study using reserpine, M3 muscarinic receptor-mediated responses might correspond to this component. Taken together, the results indicate that in addition to M2 muscarinic receptors, acetylcholine released from cholinergic intrinsic neurons might act on M3 muscarinic receptors located on the endocardial endothelium and contribute to the positive inotropic phase in cooperation with adrenergic
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pathways. Slow down of the onset of response to DMPP by atropine might be due to blockade of muscarinic receptors, but the detail mechanisms of the slowdown of onset are not clear at present. In contrast to the left atria, atropine was insensitive to the positive chronotropic/inotropic actions induced by DMPP, and the DMPPinduced action in spontaneously beating right atria of M3R-KO mice was not significantly different from that in control mice. Therefore, it is thought that a M3 muscarinic receptor-linked cholinergic mechanism is not involved in the positive chronotropic and inotropic actions of the right atria, unlike that in the case of left atria. Left and right atria-dependent different contributions of M3 muscarinic receptor function have already been reported in positive chronotropic actions of carbachol. M3 muscarinic receptor-mediated positive chronotropic actions were hardly observed in the right atria of wild-type mice, probably due to high heart rate and superior M2 muscarinic receptormediated negative chronotropic actions (Harada et al., 2012). In the right atria of rats and guinea-pigs, the presence of nonadrenergic and non-cholinergic neurons has been demonstrated by a functional study using EFS in vitro, and species-related difference in non-adrenergic, non-cholinergic innervation has been suggested (Goto et al., 1987; Saito et al., 1986). Nonadrenergic, non-cholinergic cardiac innervation has also been suggested by an in vivo study using dogs because vagal stimulation increased atrial contractility in the presence of muscarinic and b-adrenoceptor antagonists (Henning, 1992). However, in the atria of mice, we did not obtain evidence for the presence of nonadrenergic, non-cholinergic innervation because positive chronotropic and inotropic actions induced by DMPP in the right atrium were abolished by reserpine. One possible explanation is speciesrelated difference in cardiac innervation of non-adrenergic, noncholinergic neurons. Another possible explanation is difference in the methods to stimulate neural components. A ganglionic stimulant was used in the present study, but EFS exciting all neural components present in the atrial wall was used in previous studies (Goto et al., 1987; Saito et al., 1986). In the electrically stimulated left atria, atropine itself caused positive inotropic action. On the other hand, physostigmine, a cholinesterase inhibitor, elicited slowly developed negative inotropic action, which was not observed in the atria of M2R-KO mice. Atenolol caused a negative inotropic action, but the action of atenolol was abolished in atria of reserpine-treated mice. These results suggested that EFS applied to the left atria for eliciting myocardium contraction is able to stimulate cardiac cholinergic and adrenergic neurons. It should be noted that EFS-induced myocardial contraction is an integrated response of excitation of both the myocardium and cardiac interneurons. In conclusion, to clarify the functional intrinsic neurons of the mouse atrium, the effects of a ganglionic stimulant, DMPP, were characterized pharmacologically. DMPP caused biphasic inotropic/chronotropic actions in the mouse atrium. Intrinsic cholinergic neurons, acetylcholine and M2 muscarinic receptors were thought to be involved in the initial negative inotropic/chronotropic actions. On the other hand, both cholinergic and adrenergic intrinsic neural pathways were thought to mediate the following positive inotropic/chronotropic actions through activation of M3 muscarinic receptors and b1-adrenoceptors. Extrinsic neurons innervating cardiac ganglia of the mouse atrium exhibit both excitatory and inhibitory actions through activation of cardiac intrinsic neurons releasing acetylcholine or noradrenaline, and they precisely regulate the cardiac contraction.
Acknowledgments This study was partly supported by Grant-in-Aid for Scientific Research (MEXT/JSPS KAKENHI No. 22380159, Komori, S and
No. 23570081, Kitazawa, T) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
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Intramural nerve-mediated inotropic responses of left atria of rats and guinea pigs: demonstration of alpha adrenergic and nonadrenergic noncholinergic responses in guinea-pigs. J. Pharmacol. Exp. Ther. 243, 723–736. Harada, N., Ochi, K., Yaosaka, N., Teraoka, H., Hiraga, T., Iwanaga, T., Unno, T., Komori, S., Yamada, M., Kitazawa, T., 2012. Immunohistochemical and functional studies for M3 muscarinic receptors and cyclooxygenase-2 expressed in the mouse atrium. J. Auton. Pharmacol. 32, 41–52. Henning, R.J., 1992. Vagal stimulation during muscarinic and beta-adrenergic blockade increases atrial contractility and heart rate. J. Auton. Nerv. Syst. 40, 121–129. Hoard, J.L., Hoover, D.B., Wondergem, R., 2007. Phenotypic properties of adult mouse intrinsic cardiac neurons maintained in culture. Am. J. Physiol. Cell Physiol. 293, C1875–C1883. Hoard, J.L., Hoover, D.B., Mabe, A.M., Blakely, R.D., Feng, N., Paolocci, N., 2008. Cholinergic neurons of mouse intrinsic cardiac ganglia contain noradrenergic enzymes, norepinephrine transporters, and the neurotrophin receptors TrkA and p75. Neuroscience 156, 129–142. Hoover, D.B., Isaacs, E.R., Jacques, F., Hoard, J.L., Page, P., Armour, J.A., 2009. Localization of multiple neurotransmitters in surgically derived specimens of human atrial ganglia. Neuroscience 164, 1170–1179. Kitazawa, T., Asakawa, K., Nakamura, T., Teraoka, H., Unno, T., Komori, S., Yamada, M., Wess, J., 2009. M3 muscarinic receptors mediate positive inotropic responses in mouse atria: a study with muscarinic receptor knockout mice. J. Pharmacol. Exp. Ther. 330, 487–493. Levy, M.N., 1994. Vagal tachycardia In: Levy, M.N., Schwartz, P.J. (Eds.), Vagal Control of the Heart: Experimental Basis and Clinical Implications. Futura Publishing Co Inc, Armonk, NY, pp. 305–315. Levy, M.N., Martin, P., 1981. Neural regulation of the heart beat. Annu. Rev. Physiol. 43, 443–458. Loffelholz, K., Pappano, A.J., 1985. The parasympathetic neuroeffector junction of the heart. Pharmacol. Rev. 37, 1–24. ˜ o´n, M., 2006. Depletion and Martı´nez-Olivares, R., Villanueva, I., Racotta, R., Pin recovery of catecholamines in several organs of rats treated with reserpine. Auton. Neurosci. 128, 64–69. Nakatani, T., Nakashima, T., Satoh, H., 1994. Negative chronotropic and inotropic responses to nicotine in rat right and left atria. Gen. Pharmacol. 25, 865–873. Ren, L.M., Furukawa, Y., Karasawa, Y., Murakami, M., Takei, M., Narita, M., Chiba, S., 1991. Effects of tetrodotoxin and imipramine on the cardiac responses to nicotine in isolated, blood-perfused canine heart preparations. J. Cardiovasc. Pharmacol. 18, 77–84. Rysevaite, K., Saburkina, I., Pauziene, N., Vaitkevicius, R., Noujaim, S.F., Jalife, J., Pauza, D.H., 2011. Immunohistochemical characterization of the intrinsic cardiac neural plexus in whole-mount mouse heart preparations. Heart Rhythm. 8, 731–738. Saito, A., Ishikawa, T., Masaki, T., Kumura, S., Goto, K., 1986. Pharmacological analysis of autonomic innervation of the right atria of rats and guinea-pigs: demonstration of nonadrenergic noncholinergic nerves. J. Pharmacol. Exp. Ther. 238, 713–719. Struckmann, N., Schwering, S., Wiegand, S., Gschnell, A., Yamada, M., Kummer, W., Wess, J., Haberberger, R.V., 2003. Role of muscarinic receptor subtypes in the constriction of peripheral airways: studies on receptor-deficient mice. Mol. Pharmacol. 641, 1444–1451. Toda, N., Okamura, T., 2003. Pharmacology of nitric oxide in the peripheral nervous system of blood vessels. Pharmacol. Rev. 55, 271–324. Weihe, E., Schutz, B., Hartschuh, W., Anlaf, M., Schafer, M.K., Eiden, L.E., 2005. Coexpression of cholinergic and noradrenergic phenotypes in human and nonhuman autonomic nervous system. J. Comp. Neurol. 492, 370–379. Westfall, T.C., Brasted, M., 1972. The mechanism of action of nicotine on adrenergic neurons in the perfused guinea-pig heart. J. Pharmacol. Exp. Ther. 182, 409–418. Wong, K.K., 1994. Cardiac response to autonomic drugs in atria of diabetic mouse. J. Pharm. Pharmacol. 46, 513–514. Yamada, M., Miyakawa, T., Duttaroy, A., Yamanaka, A., Moriguchi, T., Makita, R., Ogawa, M., Chou, C.J., Xia, B., Crawley, J.N., Felder, C.C., Deng, C.X., Wess, J., 2001. Mice lacking the M3 muscarinic acetylcholine receptor are hypophagic and lean. Nature 410, 207–212.
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
A ganglionic stimulant, 1,1-dimethyl-4-phenylpiperazinium, caused both cholinergic and adrenergic responses in the isolated mouse atrium Kenta Ochi a, Hiroki Teraoka a, Toshihiro Unno b, Sei-ichi Komori b, Masahisa Yamada c, Takio Kitazawa a,d,n a
Department of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan Laboratory of Pharmacology, Faculty of Applied Biological Science, Gifu University, Gifu 501-1193, Japan c Common Resources Group, Okinawa Institute of Science and Technology, Okinawa 904-0411, Japan d Department of Veterinary Science, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan b
a r t i c l e i n f o
abstract
Article history: Received 26 September 2012 Received in revised form 1 February 2013 Accepted 7 February 2013 Available online 24 February 2013
An isolated atrial preparation of the mouse is useful for analyzing the actions of drugs on the myocardium, autonomic neurons and endocardial endothelium. The aim of the present study was to examine the functions of intrinsic neurons of the atrium using a ganglionic stimulant, 1,1-dimethyl-4phenylpiperazinium (DMPP). DMPP (1–100 mM) caused a negative chronotropic action followed by a positive chronotropic action in spontaneously beating right atria and also caused biphasic inotropic actions consisting of initial inhibition followed by potentiation of electrical field stimulation (EFS)induced contraction in the left atria. Inotropic actions in the left atria induced by DMPP were characterized using some autonomic drugs and M2 and/or M3 muscarinic receptor knockout (M2RKO, M3R-KO and M2M3R-KO) mice. Atropine and hexamethonium decreased the initial negative inotropic actions of DMPP. In the atria from pertussis toxin-treated, M2R-KO and M2/M3R-KO mice, the negative inotropic actions were abolished. On the other hand, the following positive inotropic actions were decreased by hexamethonium, atropine and atenolol. In the atria from reserpine-treated mice, positive inotropic actions were also decreased. The positive inotropic action induced by DMPP was almost the same in M2R-KO mice but was reduced in both M3R-KO mice and M2/M3R-KO mice. In conclusion, DMPP caused biphasic inotropic/chronotropic actions in the mouse atrium through activation of intrinsic cholinergic and adrenergic neurons. M2 and M3 muscarinic receptors and b1-adrenoceptor are thought to be involved in these actions. & 2013 Elsevier B.V. All rights reserved.
Keywords: Mouse atrium Nicotinic receptor Intrinsic neurons Cholinergic action Adrenergic action
1. Introduction Cardiac functions (contraction force, heart rate and conduction velocity) are regulated by sympathetic and parasympathetic neurons. Activation of sympathetic neurons stimulates cardiac function through release of noradrenaline, while activation of parasympathetic neurons decreases cardiac contraction and heart rate through release of acetylcholine. In general, sympathetic preganglionic fibers make their synapses near the spinal cord and postganglionic fibers arising from the ganglia innervate cardiac tissue, whereas the parasympathetic nervous system has its ganglia within organs (intrinsic cardiac ganglia, Levy and Martin, 1981; Loffelholz and Pappano, 1985). Intrinsic cardiac ganglia were initially regarded as simple relay stations in parasympathetic n Corresponding author at: Department of Veterinary Science, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan. Tel.: þ81 11 388 4795; fax: þ 81 11 387 5890. E-mail address: [email protected] (T. Kitazawa).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.02.019
preganglionic cholinergic neurons to postganglionic cholinergic neurons innervating the myocardium. However, cardiac ganglia have been demonstrated to have a complex neurochemical phenotype different from the classical view of postganglionic cholinergic neurons. Morphological study indicated that some cholinergic nerve cell bodies contained noradrenergic neurons markers, such as tyrosine hydroxylase, dopamine-b-hydroxylase and noradrenaline transporters. The presence of such hybrid intrinsic cardiac neurons has been reported in several animal species (Baluk and Gabella, 1990; Hoard et al., 2007, 2008; Hoover et al., 2009; Rysevaite et al., 2011; Weihe et al., 2005). Therefore, regulation of cardiac contractility through these kinds of intrinsic neurons might be interesting, but the functional role of these neurons has not been clarified in detail. The isolated atrium is widely used to examine inotropic and chronotropic actions of bioactive substances. Using mice atria, we demonstrated that carbachol caused biphasic inotropic actions consisting of initial negative actions (M2 muscarinic receptor) followed by slowly developed positive actions (M3 muscarinic
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receptor, Kitazawa et al., 2009). M3 receptors were demonstrated to be expressed on the endocardial endothelium, and cyclooxygenase-2/prostaglandins pathways were shown to be involved in downstream of M3 receptor activation (Harada et al., 2012). Analysis of electrical field stimulation (EFS)-induced responses of isolated visceral organs has been used to identify functional innervation. For example, analysis of EFS-induced responses indicated functional innervation of the gastrointestinal tract (Burnstock, 1972). Similarly, nitrergic nerves were demonstrated in blood vessels using EFS (Toda and Okamura, 2003). EFS has also been applied to isolated atria of rats and guinea-pigs, and characterization of evoked responses suggested inhibitory cholinergic and excitatory adrenergic innervation of cardiac tissues (Goto et al., 1987; Saito et al., 1986). However, EFS can excite all neural components in cardiac tissues (intrinsic neurons, efferent and sensory neurons) comprehensively and, therefore, EFS might not be a suitable stimulation for analyzing the function of intrinsic neurons in the mouse heart. Effects of ganglionic stimulants on the heart rate of rodents in vitro have been reported. However, the responses have not been characterized in detail (Nakatani et al., 1994; Wong, 1994). The present study was designed to elucidate the function of cardiac intrinsic neurons in the mouse atrium. To accomplish the objectives, intrinsic neurons expressing the nicotinic receptor were excited by a ganglionic stimulant, 1,1-dimethyl-4-phenylpiperazinium (DMPP), and the evoked mechanical actions were characterized. Atria from reserpine- or pertussis toxin-treated mice and muscarinic receptor knockout mice were also used for further characterization of the DMPP-induced responses.
2. Materials and methods 2.1. Animals and tissue preparations All experiments were performed in accordance with the institutional guidelines approved by the Animal Ethics Committee of Rakuno Gakuen University, Ebetsu, Hokkaido, Japan. Wild-type mice (DDY strain; Sankyo Lab Service, Sapporo, Japan) and mice lacking either the M2 muscarinic receptor (M2R-KO) or the M3 muscarinic receptor (M3R-KO) or both M2 and M3 muscarinic receptors (M2/M3R-KO) were used in the present experiments (either male or female). The generation of M2R-KO, M3R-KO and M2/M3R-KO mice has been described previously (Gomeza et al., 1999; Struckmann et al., 2003; Yamada et al., 2001). The genetic backgrounds of the mice used in the present study were 129J1 (50%) CF1 (50%) for M2R-KO mice, 129vEv (50%) CF1 (50%) for M3R-KO mice, and 129J1 (25%) 129SvEv (25%) CF1 (50%) for M2/M3R-KO mice. The animals were housed in ventilated-polycarbonate cages. The temperature of the animal room was maintained at 2371 1C with relative humidity of 40%–60% and a daily light/dark cycle (7:00 am–7:00 pm). Food (CRF-1, Oriental Yeast Co Ltd, Japan) and water were given ad libitum. Adult mice that were more than 3 months old (weight: 23–30 g) were killed by cervical dislocation. The beating heart was isolated from each animal and immersed in warmed and bubbling Krebs solution (NaCl, 118 mM; KCl, 4.75 mM; MgSO4, 1.2 mM; KH2PO4, 1.2 mM; CaCl2, 2.5 mM; NaHCO2, 25 mM; and glucose, 11.5 mM, 37 1C, gassed with 95% O2 þ5% CO2). To induce myocardial contraction, the left atrium was placed between a pair of platinum rod electrodes and suspended vertically in an organ bath. The end of the preparation was tied and connected to a force–displacement transducer. EFS (1 Hz, 2 ms in duration, 1.5 threshold voltage; Kitazawa et al., 2009; Harada et al., 2012) was applied by an electrical stimulator. After steady EFS-induced
contraction had been established, several drugs including DMPP were applied to the organ bath and the evoked responses were observed. In experiments for observation of chronotropic action, one end of the beating right atrium was attached with thread to a stationary glass rod, and the opposite end was tied with thread to a force–displacement transducer to record spontaneous contraction. 2.2. Experimental protocols Inotropic actions: After establishment of steady EFS-induced contraction (generally 60–70 min of equilibration time) of the left atrium, DMPP (1–100 mM) was added to the organ bath and its effects on EFS-induced contraction was observed for 15 min. After determining the concentration–response relationships of DMPP, pharmacological properties of the DMPP-induced inotropic actions were characterized using several autonomic drugs. Since the inotropic actions induced by DMPP (100 mM) were not reproducible even at 1-h-interval application in this experimental condition, DMPP was applied only one time in one preparation. In brief, atrial preparations were treated with respective autonomic drugs for 15 min and then DMPP was applied and its inotropic action was compared with that in non-treated atria (control). To examine the involvement of the M2 muscarinic receptor in DMPPinduced actions, DDY mice were treated with pertussis toxin (300 mg/kg, i.p.) 96 h prior to experiments. Pertussis toxin has been shown to abolish M2 receptor-mediated negative chronotropic and inotropic actions in the mouse atrium though inhibition of Gi protein function (Kitazawa et al., 2009). Treatment with pertussis toxin did not cause any obvious behavioral changes or toxic effects. After 96 h, atria were isolated and the inotropic actions induced by DMPP were compared with those in control atria from non-treated mice. Involvement of adrenergic neurons in DMPP-induced actions was examined using atria from reserpine-treated mice. Reserpine inhibits adrenergic neural function through depletion of noradrenaline contents (Martı´nezOlivares et al., 2006). Reserpine was injected (10 mg/kg, i.p.) and atria were isolated 18 h later, and the effects of DMPP were compared with the control. Catalepsy was observed in all mice treated with reserpine. DMPP-induced inotropic actions were also examined in atria from muscarinic receptor knockout mice (M2RKO, M3R-KO and M2/M3R-KO mice), and muscarinic receptor subtypes involved in the actions were characterized. Chronotropic actions: Spontaneously beating right atria from control (non-treated) mice, pertussis toxin-treated mice, reserpinetreated mice and muscarinic receptor knockout mice were suspended vertically in an organ bath and equilibrated for 1 h. DMPP (1–100 mM) was applied, and its effects on both heart rate and contraction amplitude were analyzed as chronotropic and inotropic actions. Similar to the case of the left atrium, the DMPP-induced chronotropic action at 100 mM was not reproducible at 1-h-interval application. Therefore, 100 mM DMPP was applied one time in each atrium for characterization of pharmacological properties. In brief, atrial preparations were treated with respective autonomic drugs for 15 min and then DMPP (100 mM) was applied to compare the induced actions in the presence and absence of drugs. 2.3. Chemicals The following chemicals were used: Atenolol (Sigma), atropine sulfate (Wako Pure Chemicals, Osaka, Japan), 1,1-dimethyl-4phenylpiperazinium iodide (DMPP, Sigma), hexamethonium chloride dehydrate (Wako), physostigmine sulfate (Wako), reserpine (Apoplon, Daiichi Sankyo, Tokyo, Japan) and pertussis toxin (Wako). Pertussis toxin was dissolved in sterilized saline and was
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Fig. 1. Effects of various concentrations of DMPP on heart rate and amplitude of contraction in spontaneously beating right atria of mice. DMPP (1, 10, 30, 100 mM) was applied to an organ bath in which a spontaneously beating right atrium was suspended, and changes in heart rate (A, B) and contraction amplitude (C, D) were examined. Initial negative chronotropic (B) and negative inotropic actions (D) observed within 60 s are highlighted. Ordinate: relative heart rate and amplitude of contraction (absence of DMPP¼ 100%). Values are mean 7 S.E.M. of more than four experiments.
given to mice (300 mg/kg, i.p.) in a volume of 0.1 ml/10 g body weight. Reserpine was also given to mice (10 mg/kg, i.p.).
3.2. Pharmacological properties of DMPP-induced actions in the right atria
2.4. Statistics
Since positive and negative inotropic/chronotropic actions in the right atria were marked at 100 mM DMPP, this concentration was used to determine the pharmacological properties. First, the effects of some autonomic drugs on the transient negative chronotropic actions were examined (Fig. 2A). Atropine (1 mM) or hexamethonium (100 mM) significantly decreased the transient negative chronotropic actions. On the other hand, atenolol (1 mM) did not change the actions. DMPP-induced actions were compared in the right atria from pertussis toxin- and reserpinetreated mice. These treatments did not affect beating rate of isolated atria (pertussis toxin: 335 712 beats/min, n ¼4; reserpine: 367735 beats/min, n ¼4). DMPP-induced negative chronotroic actions were abolished in the atria from pertussis toxintreated mice but did not change in the atria of reserpine-treated mice (Fig. 2A). In the atria of reserpine-treated mice, negative chronotropic actions lasted a long time compared with that in control mice (relative heart rates at 20 s, 30 s and 40 s after application, control: 10275.5%, 13077.6% and 13477%, n¼6; reserpine: 7378%, 8476% and 9974.5%, n¼4). In the atria from muscarinic receptor knockout mice, heart rate was not significantly different from that of control mice (M2R-KO: 335734 beats/min, n¼ 5; M3R-KO: 356733 beats/min, n¼5; M2/M3R-KO: 314737 beats/ min, n¼5). Negative chronotropic action of DMPP was abolished and, reversibly, positive chronotropic action was observed at 10 s in M2R-KO mice (189710%, n¼5) and M2/M3R-KO mice (17172.5%, n¼5). In the atria of M3R-KO mice, the DMPP-induced action (10974.3%, n¼5) was not different from that in control (wild-type) mice (Fig. 3A). Pharmacological properties of the DMPP (100 mM)-induced negative inotropic actions were examined (Fig. 2C). The transient negative inotropic actions were inhibited significantly by atropine (1 mM) and hexamethonium (100 mM). However atenolol (1 mM)
Data are expressed as means7 S.E.M of at least four independent experiments using left and right atria isolated from different mice. Students’ t-test or one-way analysis of variance (ANOVA) followed by Dunnett’s test was used for analysis of the effects of drugs on actions of DMPP. One-way ANOVA followed by Bonferroni’s multiple comparison test was used to compare the actions of DMPP among muscarinic receptor knockout mice. Po0.05 was considered to be statistically significant.
3. Results 3.1. Effects of DMPP in spontaneously beating right atria Spontaneous contraction of the right atria was 351720 beatsmin (n¼ 8) in the present experiments. The effects of DMPP on heart rate and amplitude of spontaneous contraction are shown in Fig. 1. One mM DMPP did not cause any change in heart rate or amplitude of contraction. However, higher concentrations of DMPP (10–100 mM) increased heart rate in a concentrationdependent manner, and the positive chronotropic actions lasted for up to 5 min. However, when 100 mM DMPP was applied, a transient decrease in heart rate (relative heart rate¼9171.8%, n ¼6) was observed from 5 to 15 s after application (Fig. 1B). DMPP (10–100 mM) also affected the amplitude of spontaneous contraction. Ten mM DMPP caused only small positive inotropic actions, but 30 or 100 mM DMPP caused transient negative inotropic actions (at 10 s, relative amplitude, 30 mM¼80 79.3%, n ¼5; 100 mM¼7476.6%, n¼5) followed by positive inotropic actions (Fig. 1C and D).
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Fig. 2. Pharmacological properties of DMPP (100 mM)-induced actions in spontaneously beating right atria. DMPP-induced chronotropic actions were divided into initial negative actions (A) and following positive actions (B). DMPP-induced inotropic actions were also divided into initial negative actions (C) and following positive actions (D). Effects of hexamethonium (C6, 100 mM), atropine (1 mM) and atenolol (1 mM) on DMPP-induced actions were investigated. DMPP-induced actions were also observed in the right atria from pertussis toxin (PTX)- and reserpine-treated mice. Each column indicates relative values of heart rate (chronotropic action, A, B) and contraction amplitude (inotropic action, C, D). Values are means7 S.E.M. of more than four experiments. *Po 0.05, **Po 0.01. Significantly different from the control response of DMPP (black column).
did not change the actions. The negative inotropic actions were not decreased but tended to be potentiated in the atria of reserpine-treated mice. In the atria of pertussis toxin-treated mice, the inhibition of amplitude by DMPP was abolished (Fig. 2C). Comparison of the inotropic actions induced by DMPP in the muscarinic receptor knockout mice indicated that the negative inotropic action was unchanged in the atria from M3R-KO mice (80716%, n ¼5) but significantly decreased and changed to positive inotropic responses in the atria from M2R-KO mice (129711%, n ¼5) and M2/M3R-KO mice (12075.7%, n ¼5) (Fig. 3B). The DMPP-induced positive chronotropic and positive inotropic actions were also characterized. On the basis of the time course of DMPP-induced responses, chronotropic action was determined at 60 s and inotropic action was determined at 45 s after application (Fig. 1A and C). Positive chronotropic actions were not changed by atropine but were significantly decreased by hexamethonium (100 mM) and atenolol (1 mM). In the atria from reserpine-treated mice, positive chronotropic actions of DMPP were abolished, but pertussis toxin treatment was ineffective in decreasing the positive chronotropic actions (Fig. 2B). Among muscarinic receptor knockout mice, positive chronotropic action was significantly enhanced in the atria from M2R-KO mice (215716%, n¼5) and M2/M3R-KO mice (226 730.6%, n¼5) but was almost the same in atria from M3R-KO mice (13875.4%, n ¼5) (Fig. 3A). Next, the DMPP-induced positive inotropic actions were characterized. Relative amplitude of spontaneous contraction at 45 s was 154 77.0% (n¼5), and this action was significantly decreased by hexamethonium (100 mM) and atenolol (1 mM). However, atropine (1 mM) was ineffective in decreasing
the inotropic action. The positive inotropic action was abolished in the atria from reserpine-treated mice but was almost the same in the atria from pertussis toxin-treated mice (Fig. 2D). The DMPP-induced inotropic actions were compared among muscarinic receptor knockout mice. The relative amplitudes of spontaneous contraction at 45 s were almost the same in the atria of M2R-KO, M3R-KO and M2/M3R-KO mice: 153 719% (n ¼5), 14675.6% (n¼ 5) and 139 717.5% (n ¼5), respectively (Fig. 3B).
3.3. Effect of DMPP on EFS-induced contraction of left atria After observing the reproducible EFS-induced contraction, DMPP (1–100 mM) was applied in an organ bath and change in the amplitude of contraction was investigated. DMPP caused a transient small negative inotropic action followed by long-lasting positive inotropic action. The negative inotropic action was shortlived and was not observed after 30 s of application. The relative amplitudes of EFS-induced contraction at 15 and 30 s were 10171.3% and 10273% (1 mM, n ¼4), 100 71% and 10574.8% (10 mM, n ¼7), 96.4 71.9% and 10772.8% (30 mM, n¼9), and 9272.2% and 11777.4% (100 mM, n ¼7), respectively, indicating concentration-dependency of negative inotropic actions (Figs. 4 and 5). The time course of DMPP-induced inotropic actions indicated that the maximum responses occurred at about 3–7 min after application and that the maximum inotropic actions were 10271.4% (1 mM, n¼4), 11873.6% (10 mM, n¼7), 13076.1% (30 mM, n¼9), and 18677.4% (100 mM, n¼7). These positive inotropic responses were observed even at 15 min after application (Fig. 5).
K. Ochi et al. / European Journal of Pharmacology 704 (2013) 7–14
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Fig. 4. Effects of DMPP on EFS-induced contraction of isolated left atria of mice. Each trace indicates a typical inotropic action of DMPP (100 mM) in the absence (control) and presence of hexamethonium (C6, 100 mM), atropine (1 mM) and atenolol (1 mM). Left atria were isolated from pertussis toxin (PTX)- and reserpinetreated mice, and DMPP-induced actions were also examined. DMPP was applied in an organ bath at the time point indicated by an arrow (.).
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Time (sec) Fig. 3. Comparison of DMPP (100 mM)-induced actions on heart rate and amplitude of spontaneous contraction in right atria isolated from muscarinic receptor knockout mice. Each graph shows the time courses of DMPP (100 mM)-induced chronotropic action (A) and inotropic action (B) observed in wild-type (control), M2R-KO, M3R-KO and M2/M3R-KO mice. Ordinate: relative heart rate and amplitude of spontaneous contraction (absence of DMPP ¼100%). Statistical comparison was performed as described in the text (see results). Values are means7 S.E.M. of more than four experiments.
3.4. Pharmacological properties of DMPP-induced actions in the left atria Negative inotropic actions of DMPP (100 mM) were decreased by treatment with atropine (1 mM) or hexamethonium (100 mM) but tended to be enhanced by atenolol (1 mM). In the atria from pertussis toxin-treated mice, DMPP-induced negative inotropic action was decreased significantly (Figs. 4 and 6A). Next, the maximum positive inotropic responses were compared in the absence and presence of autonomic drugs. The positive inotropic action of DMPP was reduced by atropine (1 mM) or hexamethonium (100 mM) through decreasing onset of the responses. Atenolol (1 mM) and reserpine-treatment were also effective in decreasing the positive inotropic actions. Inhibition of the positive inotropic actions by reserpine was partial and different from the case in the right atria (Figs. 4 and 6B). The inotropic actions induced by DMPP were compared in the atria from muscarinic receptor knockout mice (Figs. 7 and 8). The initial inhibition of EFS-induced contraction was abolished in the atria from M2R-KO and M2/M3R-KO mice. The relative
amplitudes of EFS-induced contraction at 15 s were 10773% (n¼7) in M2R-KO mice, 9471.8% (n¼4) in M3R-KO mice and 10071.1% (n¼6) in M2/M3R-KO mice. On the other hand, positive inotropic actions were significantly decreased in the atria from M3RKO and M2/M3R-KO mice but not in the atria from M2R-KO mice. A significant difference was also found between the values of M2R-KO mice and M2/M3R-KO mice (Figs. 7 and 8).
3.5. Inotropic actions of atropine, physostigmine and atenolol During the present experiments, it was observed that atropine itself caused slight changes in amplitude of the EFS-induced contraction. It was speculated that intrinsic nerves were also stimulated by EFS. Therefore, we examined the effects of cholinergic and adrenergic drugs on the EFS-induced contraction. Atropine (1 mM) slowly potentiated EFS-induced contraction, and the response reached a peak after 5 min of application (12174.6%, n ¼6). On the other hand, physostigmine (300 nM) decreased the EFS-induced contraction. Relative amplitude of EFS-induced contraction was 60.874.1% (n ¼5) at 10 min after application, but physostigimine-induced inhibition was not observed in the atria from M2R-KO mice (9872.0%, n¼5). Atenolol (1 mM) was effective in decreasing the EFS-induced contraction. Inhibition developed slowly and reached a peak after 12 min of application (relative contraction ¼8672.4%, n ¼4), but in the atria from reserpine-treated mice, atenolol-induced inhibition disappeared (relative contraction¼ 9671.5%, n ¼4, p¼0.006 vs. control).
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Fig. 5. Concentration-dependent inotropic actions by DMPP in the left atria of mice. DMPP (1, 10, 30, 100 mM) was applied to an organ bath, and time courses of inotropic actions are indicated on a long time scale (A, 0–15 min) and short time scale (B, 0–1 min, for clarification of negative inotropic action). Ordinate: relative amplitude of contraction (absence of DMPP¼100%). Values are means7 S.E.M. of more than four experiments.
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Fig. 6. Pharmacological properties of DMPP (100 mM)-induced negative (A) and positive inotropic actions (B) in the left atria of mice. Effects of hexamethonium (C6, 100 mM), atropine (1 mM) and atenolol (1 mM) on DMPP-induced actions were investigated. DMPP-induced inotropic actions were also examined in the left atria from pertussis toxin (PTX)- and reserpine-treated mice. Each column indicates relative amplitude of EFS-induced atrial contraction (absence of DMPP ¼ 100%). Values are means7 S.E.M. of more than 4 experiments. *P o0.05, **P o0.01. Significantly different from the control response of DMPP (black column).
4. Discussion Immunohistochemical studies have revealed the neurochemical characteristics of cardiac intrinsic neurons of the mouse heart, and cholinergic neurons and cholinergic neurons containing adrenergic neural markers (tyrosine hydroxylase, dopamine-b-hydroxylase and noradrenaline transporters) have also been demonstrated (Hoard et al., 2007, 2008; Rysevaite et al., 2011). However, there have been few functional studies on these intrinsic cardiac neurons, although mice have been widely used in physiological and pharmacological studies on cardiac function, and genetically manipulated mice are available for use. This functional study showed the characteristics of cardiac intrinsic neurons in the mouse atrium. DMPP, a ganglionic stimulant, caused biphasic inotropic/chronotropic actions consisting of both cholinergic and adrenergic properties, and M2 muscarinic receptor, M3 muscarinic receptor and b1-adrenoceptor are thought to be involved in the actions of DMPP. Therefore, it is thought that intrinsic neurons with cholinergic and/or adrenergic neural properties have physiological roles in the regulation of cardiac contraction in mice. Studies on the effects of DMPP and nicotine on contractility of isolated atrial preparations have been carried out using rats, mice, guinea-pigs and rabbits (Cheav et al., 1992; Nakatani et al., 1994; Westfall and Brasted, 1972; Wong, 1994). Negative inotropic/chronotropic actions (Nakatani et al., 1994) and positive inotropic actions (Cheav et al., 1992; Westfall and Brasted, 1972; Wong, 1994) have been reported as responses to ganglionic stimulants. Adrenergic neurons were shown to be involved in the positive inotropic actions
(Cheav et al., 1992; Westfall and Brasted, 1972), but negative inotropic actions were not modified by atropine or hexamethonium and were thought to be the result of direct action on cardiac muscle cells (Nakatani et al., 1994). In an isolated blood perfused canine right atrium, nicotine caused negative chronotropic actions followed by positive chronotropic actions (Ren et al., 1991). In the isolated atria of mice, DMPP caused biphasic actions consisting of initial negative chronotropic/inotropic actions followed by long-lasting positive chronotropic/inotropic actions similar to previous results obtained in the canine right atrium (Ren et al., 1991). Negative chronotropic and inotropic actions were inhibited by hexamethonium and atropine, and these actions were not observed in the atria from pertussis toxin-treated or M2R-KO mice. Since negative inotropic/chronotropic actions of carbachol were abolished in the atria of pertussis toxintreated and M2R-KO mice (Kitazawa et al., 2009; Harada et al., 2012), the results of the present study suggested that DMPP excited nicotinic receptors of intrinsic cholinergic neurons and that released acetylcholine reduced atrial contractility through activation of M2 muscarinic receptors located on the myocardium (Harada et al., 2012). Mouse cardiac ganglia containing intrinsic cholinergic neurons have already been demonstrated morphologically, and acetylcholine elicited a hexamethonium-sensitive inward current in cholinergic neurons (Hoard et al., 2007, 2008; Rysevaite et al., 2011). Taken together, these results suggest that the physiological function of these cholinergic intrinsic neurons is to decrease heart rate and contraction. In a previous study on the mouse atrium, negative chronotropic/inotropic actions were not found (Wong, 1994). Since negative chronotropic/ inotropic actions of DMPP are weak and short-lived and are soon
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overcome by following positive chronotropic/inotropic actions, it is possible that transient negative responses were overlooked in the previous study. DMPP caused positive chronotropic actions in the right atria and positive inotropic actions in both atria, and these actions were decreased by hexamethonium, atenolol and reserpine-treatment. Since atenolol is a blocker of the cardiac b1-adrenoceptor and reserpine is an adrenergic neuron blocker, the present results indicated the involvement of adrenergic neurons in positive chronotropic/inotropic actions, consistent with the results of previous studies (Cheav et al., 1992; Westfall and Brasted, 1972). However, involvement of adrenergic neurons in the DMPP-induced actions was thought to be different between the right and left atria, because reserpine abolished the positive chronotropic/inotropic actions of DMPP in the right atria but only partially inhibited the actions in the left atria. Non-adrenergic mechanisms are suggested to be involved in the positive inotropic action of DMPP in the left atria. Immunohistochemical studies have demonstrated the presence of cholinergic, adrenergic and biphenotypic intrinsic neurons in mouse cardiac ganglia (Hoard et al., 2007, 2008; Rysevaite et al., 2011). Generally, in sympathetic neurons, the nicotinic receptor is thought to be located on the cell body and it causes a depolarization-eliciting action potential, which then triggers Ca2 þ -dependent exocytosis at the axon terminal (Boehm and Huck, 1997). Therefore, the results suggested that intrinsic adrenergic or biphenotypic neurons in mouse cardiac ganglia have a regulatory function and that released noradrenaline stimulates b1-adrenoceptors for positive inotropic and chronotropic actions. Parasympathetic vagus nerves are thought to have synapses in cardiac ganglia (Levy and Martin, 1981; Loffelholz and Pappano, 1985). The results of the present in vitro study can explain the vagal nerve-mediated tachycardia (adrenergic neurons being partially involved) observed in an in vivo study (Levy, 1994).
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Fig. 7. Typical inotropic actions of DMPP (100 mM) in left atria isolated from wild-type (control), M2R-KO, M3R-KO and M2M3R-KO mice. Initial negative inotropic action was abolished in the atria from M2R-KO mice and M2M3R-KO mice. Amplitude of positive inotropic action was attenuated in the atria from M3R-KO and M2M3R-KO mice.
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Fig. 8. Involvement of M2 and M3 muscarinic receptors in negative and positive inotropic actions of the mouse left atrium induced by DMPP. DMPP (100 mM) was applied to an organ bath, and the evoked negative (A) and positive (B) inotropic actions (see Fig. 7) were compared. Each column indicates relative amplitude of EFS-induced atrial contraction. Values are means7 S.E.M. of more than four experiments. *P o0.05, **Po 0.01. Significantly different from the value for wildtype mice (black column). #P o0.05. Significantly different from the value for M2R-KO mice (open column).
We previously reported that M3 muscarinic receptors were involved in the carbachol-induced positive inotropic actions of the mouse left atrium through activation of the cyclooxygenase-2prostaglandin pathway (Kitazawa et al., 2009; Harada et al., 2012). In the present study, DMPP-induced inotropic action was significantly decreased in the left atria of M3R-KO and M2/M3R-KO mice but not in those of M2R-KO mice. In the atria of wild-type mice, atropine slowed down the onset of the response and inhibited the positive inotropic action of DMPP. These results indicated the involvement of M3 muscarinic receptors in the DMPP-induced positive inotropic action of the left atrium. Since a non-adrenergic positive inotropic component in the left atria was suggested to be present by the functional study using reserpine, M3 muscarinic receptor-mediated responses might correspond to this component. Taken together, the results indicate that in addition to M2 muscarinic receptors, acetylcholine released from cholinergic intrinsic neurons might act on M3 muscarinic receptors located on the endocardial endothelium and contribute to the positive inotropic phase in cooperation with adrenergic
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pathways. Slow down of the onset of response to DMPP by atropine might be due to blockade of muscarinic receptors, but the detail mechanisms of the slowdown of onset are not clear at present. In contrast to the left atria, atropine was insensitive to the positive chronotropic/inotropic actions induced by DMPP, and the DMPPinduced action in spontaneously beating right atria of M3R-KO mice was not significantly different from that in control mice. Therefore, it is thought that a M3 muscarinic receptor-linked cholinergic mechanism is not involved in the positive chronotropic and inotropic actions of the right atria, unlike that in the case of left atria. Left and right atria-dependent different contributions of M3 muscarinic receptor function have already been reported in positive chronotropic actions of carbachol. M3 muscarinic receptor-mediated positive chronotropic actions were hardly observed in the right atria of wild-type mice, probably due to high heart rate and superior M2 muscarinic receptormediated negative chronotropic actions (Harada et al., 2012). In the right atria of rats and guinea-pigs, the presence of nonadrenergic and non-cholinergic neurons has been demonstrated by a functional study using EFS in vitro, and species-related difference in non-adrenergic, non-cholinergic innervation has been suggested (Goto et al., 1987; Saito et al., 1986). Nonadrenergic, non-cholinergic cardiac innervation has also been suggested by an in vivo study using dogs because vagal stimulation increased atrial contractility in the presence of muscarinic and b-adrenoceptor antagonists (Henning, 1992). However, in the atria of mice, we did not obtain evidence for the presence of nonadrenergic, non-cholinergic innervation because positive chronotropic and inotropic actions induced by DMPP in the right atrium were abolished by reserpine. One possible explanation is speciesrelated difference in cardiac innervation of non-adrenergic, noncholinergic neurons. Another possible explanation is difference in the methods to stimulate neural components. A ganglionic stimulant was used in the present study, but EFS exciting all neural components present in the atrial wall was used in previous studies (Goto et al., 1987; Saito et al., 1986). In the electrically stimulated left atria, atropine itself caused positive inotropic action. On the other hand, physostigmine, a cholinesterase inhibitor, elicited slowly developed negative inotropic action, which was not observed in the atria of M2R-KO mice. Atenolol caused a negative inotropic action, but the action of atenolol was abolished in atria of reserpine-treated mice. These results suggested that EFS applied to the left atria for eliciting myocardium contraction is able to stimulate cardiac cholinergic and adrenergic neurons. It should be noted that EFS-induced myocardial contraction is an integrated response of excitation of both the myocardium and cardiac interneurons. In conclusion, to clarify the functional intrinsic neurons of the mouse atrium, the effects of a ganglionic stimulant, DMPP, were characterized pharmacologically. DMPP caused biphasic inotropic/chronotropic actions in the mouse atrium. Intrinsic cholinergic neurons, acetylcholine and M2 muscarinic receptors were thought to be involved in the initial negative inotropic/chronotropic actions. On the other hand, both cholinergic and adrenergic intrinsic neural pathways were thought to mediate the following positive inotropic/chronotropic actions through activation of M3 muscarinic receptors and b1-adrenoceptors. Extrinsic neurons innervating cardiac ganglia of the mouse atrium exhibit both excitatory and inhibitory actions through activation of cardiac intrinsic neurons releasing acetylcholine or noradrenaline, and they precisely regulate the cardiac contraction.
Acknowledgments This study was partly supported by Grant-in-Aid for Scientific Research (MEXT/JSPS KAKENHI No. 22380159, Komori, S and
No. 23570081, Kitazawa, T) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
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